The purpose of the present study is to develop a simple, fast and sensitive LC/MS method for simultaneous separation and the determination of an active component in the oriental medicinal mushroom Cordyceps militaris. Based on this work, the contents of cordycepin in Cordyceps militaris fruiting cultivated on various media were determined and compared. And also, the nutritional components such as minerals and vitamins were determined in order to provide useful information to consumer as a food material. The analysis methods of nutritional components were chosen on the basis of AOAC. The optimum separation for cordycepin was achieved using a solvent gradient consisting of the mixture of 0.1% formic acid in methanol (solvent B) in a background of 0.1% formic acid in water (solvent A) as a mobile phase and a 3.0×150 Waters XTera column. Selective ion monitoring (SIR) mode ([M+H]+ at m/z 252) was used for quantitative analysis of cordycepin. The cultivated Cordyceps militaris on various media contained 1~14 /g of cordycepin, 0.65~1.08% of thiamine, 0.86~7.17% of riboflavin, and 3.01~5.26% of niacin. The content of mineral components varied on categories, especially contained 500~3500% of potassium as a major mineral. Cordycepin, niacin and potassium were found much higher in the fruiting cultivated with soy power media (gold 10) than other media.