The French defender did not appear in great discomfort as he was taken off by Blancos boss Zinedine Zidane for Dani Carvajal in the 11th minute, though, with the move appearing precautionary.
An intense series of weeks will be completed with a two-legged Champions League quarter-final against Bayern Munich, and the Frenchman had hoped to keep his charges fresh ahead of their trip to Bavaria.
However, his plans on keeping Keylor Navas, Ramos and Carvajal on the bench and out of the match against minnows Alaves were dashed when Varane indicated discomfort as early as the eighth minute.
Right-back Carvajal was sent to warm up and then introduced shortly after, with Danilo moving to the opposite flank and Nacho partnering Pepe in the centre of defence.
While Alaves have almost certainly secured La Liga safety this season, they still have an outside chance of qualifying for the Europa League next season and will attempt to probe any vulnerabilites left at the back by the leaders.
Two points clear of Barcelona at the top, Madrid have almost certainly no choice but to beat Alaves given their Catalan counterparts are expected to win in Granada later on Sunday.