In this study, quantitative analysis of sclerotiorin from Penicillium sclerotiorum was established by using HPLC/colorimetry combination method at the condition of unavailability of authentic sclerotiorin compound. Mycelium was extracted with methanol, then analyzed by HPLC (a C18 reverse phase column with acetonitrile/water = 65/35, v/v as mobile phase and detected at 370 nm) and simultaneously measured OD370 with spectrophotometers. The limit of quantitation (LOQ) was 0.5 μg/mL. Recovery studies were performed using the lyophilized mycelia, with each spiked with sclerotiorin at 1 and 10 mg, respectively. The recoveries were found in the range of 97~109%. The coefficients of variation were less than 3.8% with this HPLC / OD370 combination method.