Using a combination of phosphoproteome enrichment and tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS), we robustly identified and quantified 1,833 phosphorylated proteins across three life cycle stages of Leishmania mexicana (L. mexicana) parasite. Protein kinase domain was the most enriched protein domain in the L. mexicana phosphoproteome. Additionally, this study systematically characterised the perturbing effect of HSP90 inhibition on the global phosphoproteome of the L. mexicana across its life cycle stages and showed that the inhibition causes substantially distinct molecular effects in the promastigotes and the amastigotes. While tanespimycin treatment decreased the phosphorylation of HSP90 and its co-chaperon HSP70 in the amastigotes, the opposite effect was observed in the promastigotes. Additionally, our results show that while kinase activity and microtubule motor activity are highly represented in the negatively affected phosphoproteins of the promastigotes, ribosomal proteins, protein folding, and proton channel activity are preferentially enriched in the perturbed amastigote phosphoproteome. Our results also show that RNA helicase domain was distinctively enriched among the positively affected RNA-binding amastigote phosphoproteome. This study reveals the dramatically different ways the HSP90 inhibition stress modulates the phosphoproteome of the pathogenic amastigotes and provides in-depth insight into the scope of selective molecular targeting in the therapeutically relevant amastigotes.