Leishmune vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixed L. (L.) chagasi promastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania (Leishmania) chagasi infected dogs and Leishmune vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128-1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP=20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasi infected dogs from Leishmune vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP <or=20% to exclude the diagnosis of CVL, a PPFP values higher 80%, mostly observed for infected dogs (INF) have a minimal change to come from a non-infected animal (NI) or Leishmune vaccinees (VAC). Together, our findings showed the potential of both anti-L. chagasi FC-AFPA-IgG and IgG2 to distinguish the serological reactivity of L. chagasi infected dogs from Leishmune vaccinees, which will further contribute for the differential diagnosis in the context of CVL immunoprophylaxis.