BACKGROUND: Uniform design was used to optimize the ultrasonic-assisted extraction of cordycepin from cultured Cordyceps militaris. The peak area of cordycepin identified using high performance liquid chromatography (HPLC) at a detection wavelength of 260 mn was considered the detection index. Three factors, ethanol concentration, extraction time and volume ratio of solvent to sample were studied. Optimal quadric polynomial step by step regression was applied to process the experimental results. RESULTS: Results show that the main factors affecting cordycepin extraction yield were the volume ratio of solvent to sample, extraction time and ethanol concentration, in that order. Cordycepin extraction yield reached a peak with ethanol concentration 20.21%, extraction time 101.88min, and volume ratio of solvent to sample 33.13 mL g(-1). An extraction model was developed based on the findings. CONCLUSION: A direct, reliable and accurate assay has been developed for the quantification of cordycepin in cultured Cordyceps militaris by a HPLC-DAD method. The validation procedure confirmed that this method is appropriate for the quality control of cordycepin. Results show that the uniform experimental design approach is useful for finding polynomial functions describing the relationships between variables and responses and to find the best experimental conditions for the extraction of cordycepin in the experimental domain considered. (c) 2007 Society of Chemical Industry.