Recent efforts in the study of vector-borne parasitic diseases (VBPDs) have emphasized an increased consideration for preventing drug resistance and promoting the environmental safety of drugs, from the beginning of the drug discovery pipeline. The intensive use of the few available antileishmanial drugs has led to the spreading of hyper-resistant strains, resulting in a chronic burden of the disease. In the present work, we have investigated the biochemical mechanisms of resistance to antimonials, paromomycin, and miltefosine in three drug-resistant parasitic strains from human clinical isolates, using a whole-cell mass spectrometry proteomics approach. We identified 14 differentially expressed proteins that were validated with their transcripts. Next, we employed functional association networks to identify parasite-specific proteins as potential targets for novel drug discovery studies. We used SeqAPASS analysis to predict susceptibility based on the evolutionary conservation of protein drug targets across species. MATH-domain-containing protein, adenosine triphosphate (ATP)-binding cassette B2, histone H4, calpain-like cysteine peptidase, and trypanothione reductase emerged as top candidates. Overall, this work identifies new biological targets for designing drugs to prevent the development of drug resistance, while aligning with One Health principles that emphasize the interconnected health of people, animals, and ecosystems.