Background There exist different methods for the determination of sun protection factor (SPF) values for sunscreens. Objectives We aimed to develop a new in vitro method using EBT3 Gafchromic® film as a substrate. Materials & Methods The colour of EBT3 Gafchromic® film changes when exposed to UV light. Films were covered by sunscreen preparations of different SPF values ranging from 0 to 50. Uncovered and covered films were exposed to different solar light energies and their colour change was compared. Absorbance spectra of films was measured at 633 nm using a UV-VIS spectrophotometer apparatus. Results The colour of the film darkens when ultraviolet energy increases, which means that absorbance increases with exposure time. However, when films are covered by sunscreens, the colour change is less visible and the absorbance significantly decreases with increasing SPF value. Conclusion There is a linear correlation between the absorbance of EBT3 Gafchromic® film and SPF value of sunscreens covering the film. Statistical analysis demonstrated that the SPF value of a sunscreen can be predicted using EBT3 Gafchromic® film as a substrate. This is the first report of an in vitro method based on colour change of a substrate which takes into consideration exposure time, and relates more closely to conditions of real-life. Based on these parameters, this is a reliable in vitro method for SPF testing.