Hair graying is caused by oxidative stress, mitochondrial dysfunction, and impaired melanocyte function, with reactive oxygen species disrupting melanogenic pathways such as Wnt/β-catenin and MITF. The study evaluated the in vitro effects of the proteoglycan-based preparation PP-PTKL (Pilopeptan® Woman Proteokel). Antioxidant capacity was measured by copper reduction assay and promelanogenic activity in human melanocytes with inhibition of Wnt/β-catenin by drug FH535. Anti-inflammatory effects were tested by mast cell histamine release and IL-1Ra and S100A8/9 levels by ELISA. PP-PTKL significantly increased MITF expression in a dose-dependent manner, by 43.1% at 17% concentration and by 3.5%. The preparation had a synergistic antioxidant activity almost four times higher than the sum of the components. It reduced mast cell histamine release and IL-1Ra and S100A8/9 levels versus placebo. PP-PTKL has demonstrated promelanogenic, antioxidant, and anti-inflammatory effects in vitro, with potential to prevent hair graying, but requires further in vivo and clinical studies.