A high-throughput platform for assessing the activity of synthetic or natural com-pounds on the motility and development of Haemonchus contortus larvae has been estab-lished for identifying new anthelmintic compounds active against strongylid nema-todes. This study evaluated the impact of serum supplementation on larval develop-ment, motility and survival in vitro and its implications for phenotypic compound screening. Of five blood components assessed, 7.5% sheep serum significantly enhanced larval development, motility and survival compared with the original medium (LB*), leading to the formulation of an improved medium (LBS*). Proteomic analysis revealed marked differences in protein expression in larvae cultured in LBS* versus LB*, includ-ing molecules associated with structural integrity and metabolic processes. The pheno-typic screening of 240 compounds (“Global Priority Box” from Medicines Malaria Ven-ture) using LBS* yielded results distinct from those in LB*, highlighting the effect of culture conditions on screening assessments. These findings indicate/emphasise the critical need to evaluate and optimise culture media for physiologically conditions in screening platforms, improving the reliability of anthelmintic discovery.