Screening of candidate trypanocidal compounds or factors affecting invasion of mammalian cells by the infective stages of Trypanosoma cruzi in tissue culture models has primarily involved labor-intensive microscopic counting of the parasites. A very efficient method for quantitating the inhibitory effect of antimicrobial agents or signaling pathways inhibitors on T. cruzi grown in L6E9 myoblasts was devised. This assay takes advantage of the selective incorporation of [3H]uracil into nucleic acids by replicating T. cruzi amastigotes. L6E9 rat myoblasts are submitted to gamma irradiation to inhibit their replication. Uracil uptake by uninfected cells is considerably decreased by this method. Nifurtimox, benznidazole, fexinidazole, MK-436, and megazol are drugs known to have activity against T. cruzi and were used in growth inhibition assays. The results demonstrated that [3H]uracil incorporation in the presence of different concentrations of nifurtimox and benznidazole closely correlated with the number of amastigotes per 100 myoblasts in Giemsa-stained monolayers under the conditions used. This method also has the advantage to differentiate between the effects of the compounds on the invasion and the replication steps of the infection with T. cruzi, as shown by the inhibitory effect of genistein when added in invasion assays.