Abstract A method based on optimum acid hydrolysis followed by high-performance liquid chromatography (HPLC) with diode array detection was developed for quantitative determination of bio-available nucleosides, present as purine and pyrimidine bases including adenine, cytosine, guanine, hypoxanthine, thymine and uracil, in natural and cultured Cordyceps. It was found that the optimum conditions was hydrolyzing Cordyceps sample in eight folds of pure commercial perchloric acid for 1 h at 95–100 °C. The determination was achieved by using a Zorbax SB-AQ analytical column (250 mm × 4.6 mm i.d., 5 μm) at gradient elution with diode-array detection. All calibration curves showed good linearity ( r 2 > 0.999) within test ranges. The developed method showed good repeatability for the quantification of six investigated nucleobases in Cordyceps with intra- and inter-day variations of less than 9.0 and 9.1%, respectively. The validated method was successfully applied to quantify bio-availbale nucleosides in natural and cultured Cordyceps, which is helpful to control their quality.