Background and purpose Proflavine hemisulfate solution is a fluorescence contrast agent to visualize cell nuclei using high-resolution optical imaging devices such as the high-resolution microendoscope. These devices provide real-time imaging to distinguish between normal versus neoplastic tissue. These images could be helpful for early screening of oral cancer and its precursors and to determine accurate margins of malignant tissue for ablative surgery. Extemporaneous preparation of proflavine solution for these diagnostic procedures requires preparation in batches and long-term storage to improve compounding efficiency in the pharmacy. However, there is a paucity of long-term stability data for proflavine contrast solutions. Methods The physical and chemical stability of 0.01% (10 mg/100 ml) proflavine hemisulfate solutions prepared in sterile water was determined following storage at refrigeration (4–8℃) and room temperature (23℃). Concentrations of proflavine were measured at predetermined time points up to 12 months using a validated stability-indicating high-performance liquid chromatography method. Results Proflavine solutions stored under refrigeration were physically and chemically stable for at least 12 months with concentrations ranging from 95% to 105% compared to initial concentration. However, in solutions stored at room temperature increased turbidity and particulates were observed in some of the tested vials at 9 months and 12 months with peak particle count reaching 17-fold increase compared to baseline. Solutions stored at room temperature were chemically stable up to six months (94–105%). Conclusion Proflavine solutions at concentration of 0.01% were chemically and physically stable for at least 12 months under refrigeration. The solution was chemically stable for six months when stored at room temperature. We recommend long-term storage of proflavine solutions under refrigeration prior to diagnostic procedure.