Fetal bovine serum (FBS) is widely used as a supplement in cell culture due to its complex composition, which supports cell growth, and metabolism for conducting experiments with mammalian cells in vitro. However, concerns regarding reproducibility, ethical considerations, and batch variability have prompted efforts to investigate its biochemical composition and potential alternatives, to subsidize the development of chemically defined alternatives. This study aimed to analyze the qualitative and quantitative composition of different FBS samples, including growth factors, vitamins, hormones, lipids, proteins, ions, and organic compounds. Additionally, mycoplasma contamination was assessed. FBS samples from different suppliers (Brazil, USA, and Paraguay) were evaluated before and after inactivation at 56 °C for 30 min. Results demonstrated the absence of mycoplasma in all samples. Among the 58 biochemical parameters analyzed, 20 exhibited significant variability (16–102 %) in non-inactivated samples, while 19 parameters showed variations (16–84 %) after inactivation. The highest variability was observed for luteinizing hormone and transferrin. Growth factor analysis revealed that epidermal growth factor, and insulin-like growth factor type 1 concentrations were below detection limits, whereas basic Fibroblast Growth Factor, and vascular endothelial growth factor A concentrations considerably reduced after the heat inactivation process for most of the evaluated samples. These findings highlight the heterogeneity of FBS composition, which may impact reproducibility in cell-based assays. Despite being considered a universal cell culture supplement, our results demonstrate that alternatives to FBS should be explored to improve the in vitro data quality and move towards a more ethical science in the field of new approach methodologies.