We have previously described a flow cytometry-based assay to detect anti-live Leishmania (Viania) braziliensis promastigote antibodies (FC-ALPA) with prominent performance of FC-ALPA to diagnosis American tegumentary leishmaniasis (ATL). However, the laboriousness to work with live parasites represented the major drawback for using FC-ALPA in routine clinical laboratory. Herein, we have presented an upgraded technology using fixed Leishmania (Leishmania) amazonensis promastigotes as antigen (FC-AFPA). Our data demonstrated that FC-AFPA-IgG displays outstanding performance for ATL diagnosis with high sensitivity (99%) and specificity (100%). Moreover, Likelihood Ratio indicated that positive results (LR+) has an infinite times more chance to come from ATL than from non-infected individuals (NI). Despite the high frequency of cross-reactivity with putative ATL co-endemic diseases, including visceral leishmaniasis, Chagas disease and malaria, FC-AFPA-IgG showed remarkable potential for differential diagnosis with other dermatological illnesses such as leprosy and sporotrichosis. FC-AFPA-IgG subclasses analysis revealed that LTA is characterized by IgG1>IgG3>IgG2 = IgG4 anti-L. amazonensis profiling, electing FC-AFPA-IgG1 and IgG3 with better performances to diagnosis ATL diagnosis. Additionally, FC-AFPA-IgG3 showed to be a better diagnostic tool in endemic areas for malarial disease. Despite the substantial advance to work with fixed promastigotes that contributes to its higher sensitivity, the lower specificity of FC-AFPA represented the major flaws as compared to FC-ALPA, suggesting that further improvement is still required to minimize the cross-reactivity with trypanosomatidae infections. Perspectives for using a flow cytometry multiplex based methodology to simultaneously assess anti-L. amazonensis, anti-L. chagasi and anti-Trypanosoma cruzi IgG reactivity is currently under investigation.