A rapid and sensitive analytical method based on reverse-phase high-performance liquid chromatography was first developed to simultaneously determine elsinochrome C (EC) and hypocrellin A (HA) in the submerged fermentation. The mobile phase consisted of acetonitrile–water 60:40 (v/v) with a flow-rate of 1 mL/min. The calibration curves were as follows: y = 37,625x + 249,775 for EC, y = 30,813x + 556,409 for HA and linear at the investigated concentration. The correlation coefficients (R2) were 0.9989 and 0.9998 respectively for EC and HA. The limits of detection and quantification were 175 and 585 µg/L for EC and 205 and 610 µg/L for HA. The precisions of concentration and retention times were less than 2.5 and 0.3%. The recovery of the method was greater than 95.0%. The methodology was applied to analyze simultaneously EC and HA concentrations in a submerged fermentation, and was adequate for analysis of biosynthesis of perylenequinones. The method was also amplified to separate and purify EC and HA using a semi-preparative C18 column. In addition, elsinochrome C was first identified in the submerged fermentation broth of Shiraia sp. SUPER-H168. Copyright © 2011 John Wiley & Sons, Ltd.