Speaking two days after the news of the monarch's cancer diagnosis was made public, the Prince gave a short but heartfelt speech in which he made a brief reference to his wife and father's health issues.
He said: "Good evening everyone. Thank you all for being here. And thank you to those whose hard work has made this evening possible. I'd like to take this opportunity to say thank you, also, for the kind messages of support for Catherine and for my father, especially in recent days. It means a great deal to us all. It's fair to say the past few weeks have had a rather “medical” focus. So I thought I'd come to an air ambulance function to get away from it all!
"But, seriously, we gather tonight to help as many people as possible benefit from top-class care in the silhouette of London’s Air Ambulance’s flying lifelines. As a former pilot for East Anglia Air Ambulance, I know just how vital the work of air ambulance teams across the country is, and the truly life-saving difference it can make to deliver urgent medical care wherever injury strikes.
Here in London, the current aircraft have served magnificently. But our capital city needs a new fleet. And we are Up Against Time. The clue really is in the Appeal's name. By September, we need the two new red birds - decked with the latest kit such as night vision - in our skies.
You wouldn't be here if you needed much persuading, I know. But just wait to hear Milana's story later of how the Air Ambulance, and its incredible teams, have given her a second chance at life.
"I should also take this opportunity to give a mention to my - our - fellow pilot, Tom Cruise. Tom, huge thanks for supporting us tonight. And Tom if you wouldn't mind not borrowing either of the new helicopters for the next Mission Impossible, it would be appreciated.
"We have all seen on our screens that - how can I put it - you seem to have a different take on 'normal wear and tear' to the rest of us. It's not the kind that buffs out. Anyway, an enormous thank you to all for being here tonight and for supporting such a wonderful cause. You are all, each and every one of you, lifesavers."
The royal father-of-three is patron of the charity and it's a cause close to his heart as he worked as an air ambulance pilot for the East Anglia Air Ambulance between March 2015 and July 2017, where he flew emergency medical service missions.
Last year, he opened up about the emotional toll of this intense role during a visit to the Blue Light Hub, a new operational base for South Central Ambulance Service, Thames Valley Police and Buckinghamshire Fire and Rescue Service.
He said: "I was holding everything and doing it all. There were a few times I had an armour up. I took it home with me and it went from there… I do worry about people retiring. We have to be better at managing long-term health."
He's facing another challenging time at the moment following the Princess of Wales's recent abdominal surgery and King Charles' cancer diagnosis. As the monarch undergoes treatment, the heir to the throne is set to take on additional responsibilities alongside his existing schedule.
The development comes after Kensington Palace announced William's return to official duties, just hours ahead of the revelation concerning the King's condition.
The timeline for the King's full recovery and return to public engagements remains unspecified, and it is anticipated that Kate will also maintain a low profile until after the Easter period.
However, King Charles, known for his diligent work ethic, still intends to manage his state affairs and official papers from behind the scenes, and Prime Minister Rishi Sunak confirmed on Wednesday that he and the King will continue their weekly meetings, with this week's held over the phone.
He will also continue to meet with the Privy Council every month, which can also be conducted remotely. The extent to which the King's health will affect other royal tours and engagements is yet to be determined.
In the meantime, significant occasions such as the Commonwealth Day service and the 80th anniversary of D-Day could see William taking centre stage.
On Wednesday, the Prince conducted an investiture at Windsor Castle, a routine element of his royal duties, before attending the gala. Buckingham Palace's statement on Monday about the King's health was brief yet optimistic.
It read: "His Majesty has today commenced a schedule of regular treatments, during which time he has been advised by doctors to postpone public-facing duties.
“Throughout this period, His Majesty will continue to undertake State business and official paperwork as usual. The King is grateful to his medical team for their swift intervention... He remains wholly positive about his treatment and looks forward to returning to full public duty as soon as possible."