He will now contest the Gold Cup on March 15 without a prep run.
Henderson said: "Bobs Worth is not going to be able to run on Saturday, which is very disappointing for everybody, including the owners.
"We very much wanted to run, but the good thing is it doesn't affect March in any way.
"We were just doing a routine check on Monday, which all the horses have before they run.
"There was something not quite right with him and it's just going the wrong way, rather than the right way.
"We were scoping him and there was a little something there.
"It's not wise to run at the weekend if he's not going to perform to his best.
"It wasn't crucial that he ran, but it was always the plan."
The Seven Barrows handler is unfazed by the prospect of sending Bobs Worth to the Gold Cup without the benefit of another run.
His magnificent victory in the Hennessy Gold Cup at Newbury in December was his first start since he was victorious in the RSA Chase at the Festival last March.
Henderson said: "I always felt the Denman Chase (Newbury, February 9) was too close to Cheltenham for him -- he's that sort of horse.
"Circumstances have now forced to us to go without it (a Gold Cup prep run).
"He's a very straightforward horse, but it's disappointing more than anything else."