We have previously reported a novelflowcytometric based methodology to access the reactivity of seric anti-live (FC-ALPA) andfixed (FC-AFPA)L. chagasiIgG antibodies applicable for cure assessment after specific therapy of VL. Both, FC-ALPA-IgGand FC-AFPA-IgGare promising targets to be used for early cure assessment. However, ourfinding suggested that further refinements were still required to improve the performance of FC-AFPA IgG for early cure assessment inVL. In the present investigation, we have established and evaluated the performance of FC-AFPA-IgG1/IgG2/IgG3/IgG4 aiming to increase theperformance indexof the previously reported for FC-AFPA-IgG. Thedatawas expressedaspercentageoffluorescent positiveparasites after incubationof pre-fixedL. Chagasi promastigotes with the test sera samples and additionof second-step FITC-labeled anti-human IgG subclasses conjugates. The analysis of anti-L. chagasi IgG reactivity in polled será samples from VL patients demonstrated that, before the etiological treatment, the IgG subclass profile was characterized by IgG1NIgG3 with the absence of IgG2 and IgG4 at the specifics era dilution tested. Following the establishment of specific PPFP cut-off-edges to segregate negative and positive results (PPFP of 50% for FC-AFPA-IgG1 and PPFP of 40% for FC-AFPA-IgG3), the analysis of IgG1 and IgG3 reactivity demonstrated good performance for early cure assessment in VL. The analysis of individual samples indicated that despite at 2 mAT, most treated VL patients (81%) still displayed positive results in FC-AFPA-IGg1 analysis, an increased fraction of treated patients (76%) presentednegative inFC-AFPA-IgG1 analysis at 6 mAT. Interestingly, thedata from FC-AFPA-IgG3demonstratedanoutstandingperformance of thismethod toearly cure assessment inVLwith increased frequency of treated patients displaying negative results at 2 mAT (90.5%) aswell as at 6 mAT (95.2%). The analysis of likelihood ratio (LR) further confirmed the remarkable performance of FC-AFPA-IgG3 as an early complementary biomarker useful to monitor the post-therapeutic cure in human VL.