A novel method for quantitative analysis of alkaloids in Epimedium was developed based on near-infrared reflectance spectroscopy. Content of alkaloids in nine species of Epimedium was quantified by the technique. Principal component analysis, ‘2,4,4,1’ mathematical treatment, standard normal variant and de-trending and modified partial least-squares analyses were used to establish the near-infrared model for quantifying alkaloids in Epimedium. The developed model of Epimedium alkaloids based on near-infrared spectroscopy possessed powerful predictive ability. The coefficient of determination for calibration, 1 minus variance ratio, standard error of calibration and standard error of cross-validation of calibration equation were 0.899 7, 0.845 7, 0.168 6 and 0.085 3, respectively. Alkaloid levels did not differ on analysis by the near-infrared reflectance spectroscopy method and conventional analytical method. Content of alkaloids in Epimedium samples ranged from 6.537 to 20.586 mg·g^-1 dry weight. Alkaloid content varied greatly among the different Epimedium species. The proposed method based upon near-infrared spectroscopy is a fast, convenient and nondestructive alternative to the traditional analytical method and shows great potential in research and development of alkaloids in Epimedium.