Over the past decade, growing demand from many domains (research, cosmetics, pharmaceutical industries, etc.) has given rise to significant expansion of the number of in vitro cell cultures. Despite the widespread use of fetal bovine serum, many issues remain. Among them, the whole constitution of most serums remains unknown and is subject to significant variations. Furthermore, the presence of potential contamination and xenogeny elements is challenging for clinical applications, while limited production is an obstacle to the growing demand. To circumvent these issues, a Serum-Free Medium (SFM) has been developed to culture dermal and vesical fibroblasts and their corresponding epithelial cells, namely, keratinocytes and urothelial cells. To assess the impact of SFM on these cells, proliferation, clonogenic and metabolic assays have been compared over three passages to conditions associated with the use of a classic Fetal Bovine Serum-Containing Medium (FBSCM). The results showed that the SFM enabled fibroblast and epithelial cell proliferation while maintaining a morphology, cell size and metabolism similar to those of FBSCM. SFM has repeatedly been found to be better suited for epithelial cell proliferation and clonogenicity. Fibroblasts and epithelial cells also showed more significant mitochondrial metabolism in the SFM compared to the FBSCM condition. However, the SFM may need further optimization to improve fibroblast proliferation.