With just five minutes played against Turin rivals Torino, Higuain and opposition goalkeeper Salvatore Sirigu collided heavily with each other when chasing to meet Miralem Pjanic's through ball.
The striker clutched his ankle and received treatment from Juventus' medical team, attempted to carry on but was replaced by Federico Bernardeschi shortly afterwards.
Higuain scored twice and missed two chances to complete a first-half hat-trick against Tottenham on Tuesday before Tottenham mounted a highly commendable comeback to hold the edge in the Champions League last-16 tie.
The possible injury news will come as a blow to Massimiliano Allegri ahead of a league-and-cup double-header with Atlanta facing Lazio and then travelling to Wembley for the last-16 second leg.
In more positive news for Juve, Paulo Dybala returned after nearly six weeks out with a hamstring injury with a 24-minute cameo in the second half as Torino were beaten 1-0 in the derby.