The Argentine star sat out the recent victories over Cornella and Elche due to suspension after receiving the first red card of his club career – in his 753rd match for the Blaugrana.
Messi was dismissed late on in the Spanish Supercopa defeat to Athletic Bilbao for hitting out at Asier Villalibre, but he has now returned to selection for the trip to Vallecas to play the Segunda side.
Philippe Coutinho, Ansu Fati, Gerard Pique, Sergi Roberto and Sergino Dest are all unavailable for the trip due to injuries.
Koeman told reporters on Tuesday, in quotes carried by Marca: “It is true that he had some discomfort. Nor has he been out for long. He is eager, fresh and without discomfort. To win things we need a Messi in shape and at his level.
“He really wants to play and win things. He is used to winning titles. He is a winner and wants to play every match. Tomorrow we will have a talk and I will tell them the team. We have to wait until tomorrow to find out.”
The Blaugrana defeated Cornella 2-0 last week to reach this stage while Rayo eliminated top-flight Elche in the previous round.