This study applied the proteome integral solubility alteration (PISA) assay to infer molecular targets and protein–ligand interactions of candidate nematocidal compound in Haemonchus contortus. By monitoring compound-induced shifts in protein solubility across a temperature gradient, the analysis provided proteome-wide evidence of direct or indirect binding events in both adult female worms and in vitro-exsheathed third-stage larvae (xL3s). Protein extracts were prepared under standardised conditions, incubated with each test compound at ten times the LC₅₀, subjected to temperature challenges and then processed for tandem mass tag (TMT)-based quantitative proteomics using high-resolution LC-MS/MS. Resultant data sets were analysed via MaxQuant and Perseus, with protein identifications referenced to the annotated chromosome-scale genome of H. contortus Haecon-5.