Mycophenolic Acid (MPA) is an immunosuppressant; produced by Penicillium Sp. In present study an analytical method is developed for analysis of MPA in fermentation broth by High Performance Thin Layer Chromatography (HPTLC), which has been a routine analytical technique by virtue of its advantages of low operating cost, high sample throughput and need for minimum sample clean-up. The method was carried out in TLC precoated silica gel on aluminium plate 60 F254. Various mobile phases were screened, out of which chloroform: methanol: water (9.2:0.7:0.1) gave the best separation at 220 nm by densitometric evaluation. The linearity was found to be in the range of 100-500ng with R2 value 0.997, % RSD of intraday and interday precision was found to be in the range of 1.22-2.03 and 1.23-2.81 respectively. Limit of Detection (LOD) and Limit of Quantification (LOQ) of mycophenolic acid was found to be 23.13ng/band and 77.09 ng/band respectively. Recovery of MPA obtained was found to be 98.76%; which suggest that the method is simple, accurate and reproducible. The experimental data was statistically analysed by Analysis of Variance (ANOVA) which proves that, the developed chromatographic method can be used for routine analysis of MPA.