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Selecting the primary cells in an optimal state for cultured meat production is a crucial challenge in commercializing cultured meat. We investigated the metabolomic changes in culture media according to passage numbers for indirectly assessing the state of primary cells. Pig skeletal muscle stem cells (PSCs) harvested from the biceps femoris muscles of 7-d-old crossbred pigs (Landrace × Yorkshire × Duroc, LYD) were used for cell characterization. Fresh media (FM) and spent media (SM) of PSCs during passages 1 to 3 in vitro culture were prepared for metabolomics analysis. SM was collected on the third day of proliferation for each passage of PSCs. Cell characterization analysis revealed that the proliferation rate was highest at passage 2; however, a significant loss of expression of myogenic marker genes was observed at passage 3. Based on metabolomic profiles of culture media, FM and SM groups (SM1, SM2, and SM3) were clearly separated by partial least squares-discriminant analysis. A total of seven differentially abundant metabolites (DAMs) were identified from FM and SM for each passage, based on the following criteria: P < 0.05, fold change > 1.5 or < 0.66, and a variable importance in projection score > 1.5. All seven DAMs and their interconnected metabolites might be primarily used as substrates for energy production and most of them were relatively abundant in SM3. Among the seven DAMs, the three potential biomarkers (γ-glutamyl-L-leucine, cytosine, and ketoleucine), which showed significant changes exclusively in SM3, each had an area under the curve value of 1. Therefore, monitoring the levels of these key metabolites in culture media could serve as a quality control measure for cultured meat production by enabling the indirect detection of suboptimal PSCs based on their proliferation ability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)