Per- and polyfluoroalkyl substances (PFAS), ubiquitous environmental contaminants, pose significant challenges
to ecosystems and human health. While cell cultures have emerged as new approach methodologies (NAMs) in
ecotoxicity research, metabolomics is an emerging technique used to characterize the small-molecule metabolites
present in cells and to understand their role in various biological processes. Integration of metabolomics with cell
cultures, known as cell culture metabolomics, provides a novel and robust tool to unravel the complex molecular
responses induced by PFAS exposure. In vitro testing also reduces reliance on animal testing, aligning with ethical
and regulatory imperatives. The current review summarizes key findings from recent studies utilizing cell culture
metabolomics to investigate PFAS toxicity, highlighting alterations in metabolic pathways, biomarker identification, and the potential linkages between metabolic perturbations. Additionally, the paper discusses different
types of cell cultures and metabolomics methods used for studies of environmental contaminants and particularly
PFAS. Future perspectives on the combination of metabolomics with other advanced technologies, such as singlecell metabolomics (SCM), imaging mass spectrometry (IMS), extracellular flux analysis (EFA), and multi-omics
are also explored, which offers a holistic understanding of environmental contaminants. The synthesis of current knowledge and identification of research gaps provide a foundation for future investigations that aim to
elucidate the complexities of PFAS-induced cellular responses and contribute to the development of effective
strategies for mitigating their adverse effects on human health.