Abstract: Cholesterol and tocopherols, which are important quality indicators in milk powder, are essential nutrients for the human body. Current pretreatment methods for the detection of cholesterol and four isomers of vitamin E (α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol) are based on national food safety standards, which are complicated, time-consuming, and unsuited for simultaneous measurements. Thus, developing a simple, fast, and simultaneous detection method for cholesterol and the four kinds of tocopherols is of practical significance. In this study, gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to establish qualitative and quantitative methods for the determination of cholesterol and the above mentioned four isomers of vitamin E. The sample was digested with lipase and then saponified rapidly using a potassium carbonate-ethanol system. The optimal pretreatment method was established by optimizing the enzymolysis time, saponification temperature, type and volume of the extraction solvent, and extraction time. Then, cholesterol and the four tocopherols in milk power were simultaneously determined. The results revealed a good linear relationship for cholesterol and the tocopherols in the range of 0.5-50.0 mg/L and 0.25-25.0 mg/L, respectively. The correlation coefficients (r²) were greater than 0.99; the recoveries were 76.6%-93.1%; and the relative standard deviations were 0.9%-3.3%. The limits of quantification for cholesterol and the tocopherols were 10.0 μg/100 g and 5.0 μg/100 g, respectively. The recoveries of the added standards did not fully reflect the ability of the method to decompose and extract the actual sample, especially given that the five compounds considered in this study were fat-soluble. Thus, the added standard recovery could not verify the enzymatic hydrolysis effect. In order to investigate the effectiveness of this method for actual milk powder samples, the amounts of cholesterol and the four tocopherols in infant milk powder were determined according to the national standard methods (GB 5009.82-2016, GB 5009.128-2016) and the proposed method. For each method, six sets of measurements were carried out in parallel. The cholesterol content measured by this method was slightly lower than that measured by the national standard method, while the amounts of the four tocopherols were slightly higher. There was no significant difference (p> 0.05) between the national standard method and our method on the amounts of cholesterol and the four tocopherols in milk powder. Twenty kinds of infant formula milk powder and four kinds of low-fat milk powder were randomly selected from the market, and the amounts of cholesterol and the four tocopherols were analyzed. The results showed that the amounts of cholesterol and the four tocopherols in the infant formula milk powder were higher than those in the low-fat milk powder. This method is simple, fast, sensitive, and accurate, thus meeting the detection requirements for cholesterol and tocopherols in milk powder. The findings of the study would provide a theoretical foundation for the rapid estimation of milk powder quality.