Red yeast rice (RYR) food supplements are widely used for cholesterol management due to their monacolin content, particularly monacolin K (MK), which shares the same chemical structure as lovastatin, a compound associated with several side effects. The European Food Safety Authority (EFSA) highlighted uncertainties regarding the compositional vari-ability, characterization of bioactive constituents, and safety of RYR products, concluding that a safe level of monacolin intake could not be established. In this study, twenty-seven commercial semi-processed RYR ingredients for food supplements manufacturing were investigated through a combined chemical and biological approach to characterize their composition and evaluate potential safety concerns. Targeted LC-MS/MS analyses re-vealed substantial variability in MK, monacolin hydroxy acid (MKA), and dehydromon-acolin K (DMK) levels among products. Exploratory qualitative untargeted metabolomics enabled the putative annotation of multiple secondary metabolites, including azaphilones and polyketides, highlighting differences in chemical profiles. Correlation analyses indi-cated that DMK content was associated with pigment distribution and cytotoxic responses in human hepatocellular carcinoma cells and primary human skeletal muscle cells, whereas MK and MKA showed no significant association under the tested conditions. Proteomic analyses further revealed distinct cellular responses between RYR extracts and simvastatin. These findings emphasize the importance of comprehensive chemical char-acterization and quality control strategies considering both monacolins and secondary metabolites to improve the safety assessment of RYR supplements.