The Citizens are scheduled to host Amakhosi in a PSL encounter at the Cape Town Stadium in the Mother City on Tuesday.
Chiefs will be without new South Africa international Lorenzo Gordinho, who is expected to serve his suspension.
Steve Komphela's side might also be without Khune, while Sibusiso Khumalo has already been ruled out after both players picked up injuries over the weekend.
SuperSport United defeated Amakhosi 5-3 on penalties following a 1-1 draw in the Nedbank Cup quarter-final clash on Saturday evening.
The Soweto giants have since released the following statements on their official website.
"Lorenzo Gordinho picked up the fourth yellow card against Supersport United in the last game and is suspended for this match," a statement read.
"Goalkeeper Itumeleng Khune picked up a hip injury on Saturday and the doctors are working around the clock to get him ready for the match," another statement read.
"Defender Sibusiso Khumalo was withdrawn from the traveling team due to a strained thigh muscle," a statement concluded.