The use of fetal bovine serum (FBS) in cell culture is being questioned for scientific and ethical reasons, prompting the exploration of alternative approaches. Nevertheless, the influence of FBS on cell functioning, especially in fish cells, has not been comprehensively examined. This study aims to evaluate the impact of FBS on the lipidome of PLHC-1 spheroids and investigate cellular and molecular responses to plastic additives in the presence/absence of FBS. Lipidomic analyses were conducted on PLHC-1 cell spheroids using liquid chromatography coupled with a high-resolution quadrupole time-of-flight mass spectrometer (HRMS-QToF). The removal of FBS from the culture medium for 24 h significantly changed the lipid profile of spheroids, resulting in a depletion of cholesterol esters (CEs), phosphatidylcholines (PCs) and lyso-phosphatidylcholines (LPCs), while ceramides and certain glycerophospholipids slightly increased. Additionally, the exclusion of FBS from the medium led to increased cytotoxicity caused by a mixture of plastic additives and increased lipidomic alterations, including an elevation of ceramides. This study emphasizes the protective role of serum components in fish liver spheroids against a mixture of plastic additives and underscores the importance of considering exposure conditions when studying metabolomic and lipidomic responses to toxicants.