In order to establish a method to determine 16 amino acids in Hordeum vulgare L. var. nudum Hook.f and Cordyceps militaris,pre-column derivatization RP-HPLC was used with 2,4-dinitrofluorobenzene as derivative reagent,Phenomenex Gemini-NX C₁₈ as chromatographic column,mobile phase is A:0.05 mol/L sodium acetate solution (pH = 6.4,containing 1 ‰ N,N-dimethylformamide) and B:acetonitrile-water.Solution(1∶1,V/V),gradient elution,wavelength 360 nm,flow rate 1 mL/min.The results showed that the content of glutamic acid in H. vulgare L. var. nudum was 2.58%,proline 1.08%,methionine and cysteine less than 0.05%,and the average value of 16 amino acids was 8.68%.The amino acid content of C. militaris is higher than H. vulgare L. var. nudum,which the content of glutamic acid was 3.23%,aspartic acid 242% and serine 1.60%,methionine and cysteine less than 0.05%,the total amount of 16 amino acids was 17.65%.The method is simple,stable and reliable,which can provide reference for the quality control of H. vulgare L.var. nudum and C. militaris.