The control of parasitic nematodes of humans and animals remains heavily dependent on a limited number of anthelmintic drug classes, and resistance to major classes is now widespread. Although phenotypic screening readily identifies compounds that impair worm motility or development, the intrinsic biological processes underlying chemical sensitivity in parasitic nematodes remain poorly defined. Here, we identified a hit compound with a pyridyl scaffold from a phenotypic screen against the model parasitic nematode, Haemonchus contortus, and using structure–activity optimisation we generated a potent chemical probe, WEHI-684. To uncover protein networks associated with the mechanism of action, thermal proteome profiling and time-resolved quantitative proteomics interrogated WEHI-684-induced perturbations in H. contortus. Across larval and adult stages of this major parasite of livestock, proteome integral solubility alteration (PISA) profiling revealed reproducible alterations in proteins associated with cytoskeletal organisation and intracellular trafficking, including actin- and motor-related components. Complementary quantitative proteomics identified induction of an aspartyl protease and suppression of secretory CAP family proteins. Integrated analysis of these datasets supports a model in which chemical perturbation of cytoskeletal and trafficking proteins is associated with secondary modulation of proteolytic pathways, coinciding with rapid impairment of motility. These findings indicate that linked structural and proteolytic responses contribute to chemical sensitivity in H. contortus and demonstrate how integrative proteomics can resolve organism-level responses to chemical perturbation beyond single-target paradigms.