Background: Standard protocols are lacking for the preparation of platelet lysates (PL) as an alternative to using fetal bovine serum as a cell culture supplement. This study aimed to establish optimum conditions for preparing PL for use in cell cultures. Methods: Cell density in three pooled platelet concentrates (PC) were adjusted to 1×10 12 / L and 2×10 12 /L. PL was prepared from PC by 1 to 3 freeze-thaw (FT) cycles. HaCaT cells were cultured in media supplemented with 5% or 10% PL. Cell numbers were estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Growth factors were quantified by using the Luminex 200 system (Luminex Corporation, USA). Results: Cell proliferation rates in the presence of PLs were similar when prepared from PCs of both cell densities. The rates were higher in media containing 5% PL than 10% PL when prepared by two FT cycles. Concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB/BB (PDGF-AB/BB), PDGF-AA, and epidermal growth factor (EGF) were significantly higher in PL prepared from PC with a cell density of 2×10 12 /L than 1×10 12 /L PC. However, only VEGF and PDGF-AA concentrations in PLs were correlated with HaCaT cell counts. Conclusions: The 5% PL from PC with a cell density of 1×10 12 /L prepared by two FT cy- cles treatment was the most effective condition that supported steady HaCaT cell prolifer- ation. Our finding may be useful for preparing PL-supplemented cell culture media.