The Monascus fermentation product red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK) in both its lactone (MKL) and acid (MKA) forms and the mycotoxin citrinin (CT). The yellow pigments in RMR, namely, monascin (MS) and ankaflavin (AK), have been reported to exhibit antimetastatic and antiangiogenic activities. Currently, MK and these yellow pigments are usually detected in RMR by different analytical methods that are inconvenient, expensive, and time-consuming. The goal of this study was to establish a rapid, synchronous analytical method for determination of the MKA, MKL, MS, AK, and CT levels in RMR. MKA, MKL, MS, AK, and CT were extracted by the same extraction method, then separated by RP-HPLC with a C18 column. The effluent from the column was passed through a photodiode array detector and then introduced directly into a fluorescence detector. The results showed that high recovery rates of MKA, MKL, MS, AK, and CT are possible if RMR powder is extracted with 75% ethanol (10 mL) at 80 degrees C for 30 min. With regard to the optimal conditions of the HPLC, the peaks of MKA, MKL, MS, AK, and CT can be clearly separated from any noise peaks by isocratic elution with a mobile phase comprising 0.05% trifluoroacetic acid in acetonitrile-water (62.5 + 37.5, v/v).