It comes after Ancelotti praised Mbappe's 'remarkable act of altruism' in letting Jude Bellingham take a penalty during Sunday's 2-0 victory over Getafe.
When asked about future penalty duties, Ancelotti was unconcerned with whether Mbappe or Bellingham would step up.
"Bellingham or Mbappe, one of the two, whoever's in best condition," Ancelotti said.
"This isn't a debate. There are two sides to it. Mbappe against Getafe, you might see it as insecurity, or you might see it as we do, an act of responsibility and altruism."
On Wednesday, however, the decision backfired, with Mbappe missing his second penalty in the space of a week.
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Having missed from the spot in the Champions League defeat to Liverpool the week before, on Wednesday he squandered another chance to score from 12 yards against Athletic Bilbao, with goalkeeper Julen Agirrezabala saving his attempt.
Ancelotti has since doubled down on his penalty views, adding that there are 'no changes' to who will take spot kicks.
"Our penalty shooters are Mbappe, Vini [Vinicius Junior] and Jude," said the manager. "Whoever shoots is good."
Pressure has already been mounting on the France international after Liverpool goalkeeper Caoimhin Kelleher saved his attempt in Europe, as well as Didier Deschamps's omission of him from France's latest Nations League squad.
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In the Getafe match, Mbappe, who has faced increasing scrutiny following his summer transfer from Paris Saint-Germain, deferred to Bellingham when the opportunity for a spot-kick arose.
The England midfielder successfully converted, and Mbappe added to the scoreline with a goal from open play shortly after.
Mbappe's performance this season has drawn mixed reactions, with ten goals and two assists in 20 appearances.
After the loss to Bilbao, Mbappe posted on Instagram, writing: "Bad result. A big mistake in a match where every detail counts. I take full responsibility for it.
"A difficult moment but it's the best time to change this situation and show who I am."
Despite the challenges in his side, Ancelotti remains focused on finding the best way to integrate Mbappe in the line-up, whilst also incorporating Bellingham, Vinicius Jr, and Rodrygo.
Ancelotti further praised Mbappe's team-first mentality.
"He's a player with extraordinary talent, he might be the biggest talent in football, and he puts that [at the service of] the team," he said. "I value that a lot, personally."
The Italian boss was asked whether Mbappe's deferral to Bellingham showed a signs of lacking courage and leadership, to which he responded: "I don't see it like that. It's important for the team, his teammates, all of us, within the dressing room.
"We value it as I said before, a great act of altruism. When a great talent is altruistic, you're on the right track."