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Partial least squares (PLS) method was applied to establish models with near infrared spectroscopy (NIRS) for quantitative analysis of protein content in Cordyceps militaris. Varies pretreating spectra methods were used for conversion of near infrared spectra in order to remove the noise in spectra. The pretreated spectra were applied to develop PLS quantitative analysis models respectively. Each model was optimized by selecting the most suitable number of factors. The most optimum model was selected depend on the root mean squares of calibration sets calculate by cross-validation (RMSECV) and the root mean squares of predicted sets (RMSEP). RMSECV of the optimum model was 0.0199. Using the optimum model for determination of protein content in Cordyceps militaris, RMSEP was 0.0145.