An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25-cm2 flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1∶106 to 1∶10; we defined 106 tachyzoites/ml−1 as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 106 tachyzoites/ml−1. Finally, we validated the method using a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of the unknown parasite suspension. This easy-to-use method is reproducible and can be applied to any T. gondii tachyzoite RH strain, genetically modified or not. This method is also suitable for testing promising candidates for an effective live vaccine.