Visceral Leishmaniasis is a severe form of disease that is nearly always fatal in the majority of untreated cases. In the parasite the apparent dependency of synthesis and regulation of trypanothione on thiol metabolic pathway, the role of TryS and TryR in drug resistance and redox homeostasis and its interaction has remained unexplored. So, in this study, we have characterized TryS and TryR proteins of L. donovani, investigate potential interaction between them and tried to identify their correlation with oxidative stress and drug resistance. The enzymatic activities and kinetic properties of both enzymes were also studied. We elucidate for the first time the interaction between TryR and TryS in L. donovani by in silico analysis coupled with in vitro and in vivo interaction which suggests a stable complex formation between TryR & TryS in L. donovani. Further, we observed that LdTryS and LdTryR significantly upregulated at both transcriptional and translational level in Amp B resistant isolates and during transformation from proliferative logarithmic growth phase to infective stationary phase, thus help the parasite for the survival in the macrophage.