Quantification of sun-related changes in conjunctival ultraviolet autofluorescence (CUVAF) images is a subjective and tedious task, in which reproducibility of results is difficult. Thus, we have developed a semiautomatic method in MATLAB® to analyze CUVAF images retrospectively. The algorithm was validated on 200 images from 50 randomly selected participants from the Western Australian Pregnancy Cohort (Raine) study 20-year follow-up assessment, in which CUVAF area measurements were available from previous manual analysis. Algorithm performance was compared to manual measurements and yielded better than 95% correspondence in both intra- and interobserver agreement. Furthermore, the semiautomatic method significantly reduced analysis time by 50%.