The two men were hurt during the first half of Tuesday night's 2-0 EFL Cup second-round victory over Cheltenham with Mitrovic leaving the pitch before the break and replacement Gayle following him after it.
Manager Rafael Benitez would have loved to have both available for a key game, with Mitrovic having only just returned from a four-game ban, but the club will follow Football Association protocol and not allow them to return until six days after their respective head injuries.
The club's head of sports medicine and science Paul Catterson said: "The treatment and management of concussion in sport has received deserved attention in recent years.
"We have to adhere to the guidelines set out by the FA in line with other competitive sports, which rightly puts the safety and health of players first."
Benitez will have to shuffle his pack once again, although he will at least be able to call upon Ayoze Perez, whose double against Cheltenham opened his account for the season.