This study investigates the efficacy of a newly developed humanized serum-free media (SFM) that replaces Fetal Bovine Serum (FBS), in culturing of common in vitro models (Caco-2 and 3D Primary Human Hepatocytes (PHH)). The research’s focus aligns with the 3R principles (replacement, reduction, refinement) and may potentially lead to enhancing the human relevance of the in vitro models and ultimately contribute to more accurate and successful drug development in the future. This study is divided into two subprojects, Caco-2 and 3D PHH, respectively. For Caco-2, we evaluated cell morphology, proliferation and monolayer formation by assessing the permeability of marker compounds. For PHH, we investigated spheroid formation and viability using an ATP measurement assay, lipid concentration using an Adipored Assay and CYP activity of the 3D PHH. Finally, global proteomics analysis was performed for both Caco-2 cells and 3D PHH grown in SFM and in conventional medium (CM). Our findings show that Caco-2 cells could proliferate well enough in the SFMCaco-2 without any change in their cell morphology, whereas their monolayer tightness was found to be similar in SFMCaco-2 as compared to CMCaco-2. Furthermore, from the proteomics analysis there was no major difference detected between cells in SFMCaco-2 and in CMCaco-2, however several of the relevant drug transporters of this model were found in higher levels in Caco-2 cultured in SFMCaco-2. For 3D PHH, our findings indicate that spheroids could be perfectly formed in both SFM3D PHH and in CM3D PHH, having relatable viability and lipid concentration, especially after two week of culturing period. Finally, the proteomics analysis revealed no significant difference between the proteome of the spheroids when cultured in SFM3D PHH or in CM3D PHH. In conclusion, these results suggest that the SFM provides a viable and sometimes superior alternative to CM for both Caco-2 and 3D PHH, promoting the 3R principles and potentially offering a more human-relevant model for in vitro studies.