Purpose: The purpose of this study is to assess the stability of the growth factors (GF) in autologous serum eyedrops under different storage conditions.Methods: The concentration of epidermal growth factor (EGF), transforming growth factor-β (TGF-β1), platelet-derived growth factor AB (PDGF-AB), and albumin was measured in fresh and defrosted samples of autologous serum under different storage conditions. The fresh and defrosted samples were cooled at 4 °C, and they were studied immediately after preparation, or after defrosting, and after 1, 2, 3, and 4 weeks. The concentration of GF was also assessed after 1, 3, 6, and 9 months at −20 °C. We also investigated how the different storage conditions influence the biological effects of autologous serum on conjunctival and corneal cell cultures.Results: The concentration of EGF, TGF-β1, PDGF-AB, and albumin remained stable over the 4 weeks at 4 °C, both in fresh and in defrosted samples. Likewise, no statistically significant differences were found between the GF concentration in fresh samples and after 1, 3, 6, and 9 months of freezing at −20 °C. Moreover, no differences were found on the cell proliferation and differentiation between cultured cells with fresh or defrosted samples after 4 weeks at 4 °C or after 1, 3, 6, or 9 months at −20 °C.Conclusions: Long-term storage of autologous serum eyedrops at −20 °C does not affect the concentration of GF, simplifies clinical logistics, and reduces the frequency of blood extractions from the patients.