The American trypanosomiasis better known as Chagas disease is a systemic infection caused by the protozoan Trypanosoma cruzi. It is estimated 56,000 new cases and 12,000 deaths annually in the Americas region in 21 countries. The chemotherapy of the disease is still inadequate. Several proteases involving in vital processes for the survival of parasites such as invasion, migration, nutrition, immune response evasion and immunomodulation have been described. The presence of the leucyl aminopeptidase (LAPTc) in the three life stages of the parasite indicates its importance for one or several physiological processes. The objective of this work is to develop LAPTc enzymatic assays for screening compounds. Using water soluble peptide substrate Leu-Ser-Thr-Val-Ile-Val-Arg (LSTVIVR) as substrate, the RapidFire Mass Spectrometry assay provides higher signal to blank ratio, better assay performance than biochemical assay with chromogenic Leucine-p-nitroanilide (Leu-pNA) substrate and fluorogenic Leucine-7-amino-4-methylcoumarin (Leu-AMC) substrate. With this method was identified a tetrazole-peptidomimetic as a micromolar LAPTc inhibitor, with potentialities as an in vitro antichagasic agent.