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Mycophenolic acid is a secondary metabolite of Penicillium brevicompactum and has been extensively studied for its antineoplastic, immunosuppressive, anti-inflammatory, antiviral, antipsoriasis, and antifungal activities. In the present study, a high-performance thin-layer chromatography method was developed for determining mycophenolic acid and a chitosan-linked prodrug using 60 F254 silica gel plates. The mobile phase used was chloroform–methanol–water (9.2 : 0.7 : 0.1, v/v/v), which provided good separation. The linearity was found to be in the range of 4–12 µg/spot with an R2 value of 0.9973. The percentage of RSD for intra-day and inter-day precision was found to be in the range of 0.7–1.1 and 0.9–2.0, respectively. The limit of detection and limit of quantification of the prodrug were determined to be 0.27 and 0.82 µg/spot, respectively. The recovery of the prodrug obtained was found to be 99.79%, suggesting that the method is simple, accurate, and reproducible. The proposed method proved to be sensitive, accurate, precise, economical, and less time-consuming, making it suitable for successful analysis in microsphere formulations. [ABSTRACT FROM AUTHOR]