The 31-year-old was absent as Everton welcomed Newcastle to Hill Dickinson Stadium for Thursday's fourth-round tie, with the club confirming that his omission was disciplinary rather than injury-related.
David Moyes' side progressed to the quarter-finals with a 3-1 victory, but the circumstances surrounding Grealish's absence have raised questions about his immediate future at the club.
Everton announced the decision through their official team news before kick-off.
"Jarrad Branthwaite remains sidelined for a second successive game due to concussion protocols, while Jack Grealish is unavailable for selection due to a breach of Club discipline," the club stated.
According to reports, Grealish failed to attend training on Monday without authorisation.
The England international had missed Everton's previous Premier League fixture against Arsenal through illness, with the Toffees losing 2-0 at the Emirates Stadium.
He subsequently returned to training at the club's Finch Farm complex, but his unauthorised absence led to disciplinary action and the prospect of a financial penalty.
The incident is particularly unfortunate for Grealish given the importance Moyes has placed on his experience and leadership since bringing him back to Merseyside.
The winger enjoyed a productive loan spell at Everton last season, registering two goals and six assists in 20 appearances before a foot injury brought his campaign to an early conclusion.
Manchester City subsequently agreed to another season-long loan, providing Grealish with an opportunity to rebuild his fitness and rediscover the form that made him one of English football's most influential attacking players.
Moyes has repeatedly emphasised that he expects more than individual performances from the former Aston Villa midfielder.
Speaking earlier in the season, the Everton manager explained that Grealish had a responsibility to become a senior figure within a developing squad.
"I think Jack knows what I expect now. We had lots of conversations last year. He knows when he comes here there is an opportunity for him to play and to show what he can do.
"But I also want him to be a senior professional and a role model. We've got Tyrique and Tyler and if ever you are looking at somebody for how to play as a wide player, Jack Grealish would certainly be one of them.
"I think both of them could learn an awful lot from Jack and I want him to become that senior citizen amongst them, somebody who teaches them."
Moyes was referring to young attacking players Tyrique George and Tyler Dibling, with the manager keen for Grealish to use his experience to help their development.
He also believes the winger has the personality to handle the expectations that come with being one of Everton's most recognisable players.
"If there is one person who would be able to carry that [pressure] it would be Jack, just because of the way he is. He will find it easy to carry expectation and he'll be happy to do so. That's his character," Moyes said.
The manager also acknowledged that supporters were expecting Grealish to rediscover his best football during his second season at the club.
"I do think that is the case [that there is more expectation in the second season] because everybody wants Jack to get back to where he was. His first three months for us last season were hugely important in getting us wins, so now it is about getting him right to perform at that level again."
Moyes previously highlighted the physical difficulties Grealish faced following his lengthy absence, explaining that the player had spent six-and-a-half months out and had not enjoyed a full pre-season.
He also suggested that regular matches would be particularly important in helping the winger regain his sharpness.
Grealish had made six appearances for Everton this season before the Newcastle fixture, having returned to the side after his absence against Arsenal.
The disciplinary incident now provides an unwelcome complication for a player who needs consistent football to rebuild his confidence and fitness.
For Moyes, however, the message is clear: Grealish's experience and reputation bring responsibilities, and his position within the squad depends on meeting the standards expected of a senior professional.