The rhizomes of Anemarrhena asphodeloides Bunge (Liliaceae) are prescribed as crude drugs in herbal medication for the treatment of various diseases such as diabetes, inflammation, and platelet aggregation inhibition. A simple, sensitive, and precise reversed-phase liquid chromatographic method was developed to study the quantitative determination of 5 bioactive compounds from these rhizomes, namely, neomangiferin, mangiferin, isomangiferin, nyasol, and methylnyasol. Chromatographic analysis was performed on Capcell Pak C 18 column (150 × 4.6 mm, 3 μm) with a mobile phase consisting of acetonitrile, methanol, and 0.1% formic acid at a flow rate of 1.00 mL/min. Quantitation was performed using a UV-visible detector at 260 nm. The method for the determination of reported medicinal agents was accurate and reproducible. Excellent linear behavior was observed over the investigated concentration range of 2.5-100.0 μg/mLfor neomangiferin; 1.5-60.0 μg/mL for mangiferin; 0.5-20.0 μg/mL for nyasol; and 0.2-20.0 μg/mL for methylnyasol; correlation coefficient >0.99. The intraday and interday precision over the concentration range of compounds was <6.6% (relative standard deviation) and accuracy was between 94.9 and 109.3%. This method can be successfully applied for the analysis of medicinal compounds from the ethanolic extract of A. asphodeloides Bunge.