Answer: When he saw Miguel bleed
Like everyone else in the city, Tzekel-Kan believed that Miguel and Tulio were gods. However, they did not live up to his expectations - they did not want to sacrifice an El Dorado man to Xibalba, and they were clearly inferior at playing the ball game the gods had supposedly invented. Miguel in particular was more compassionate than vengeful, as Tzekel-Kan expected a god to be.
Tzekel-Kan finally had his suspicions confirmed when he made Miguel and Tulio play ball. Tulio accidentally hit Miguel in the face, which made him bleed. Tzekel-Kan saw this, and knew they were not gods. Later in the film, Tzekel-Kan said, "Gods don't bleed."