Abstract: About 1 g of evenly mixed health food sample was taken, and mixed with about 5 mL of warm water (50 ℃) and 5 mL of phosphate buffer solution (pH 7.0). After mixing well, 0.3 g of lipase and 0.5 g of papain were added, and the mixture was mixed by cover vortex for 2-3 min, and shaken at 37 ℃ for 120 min. Then 10 mL of anhydrous ethanol and 1 g of potassium carbonate were added into the above solution, and 10 mL of n-hexane and 10 mL of water were added after mixing well. The mixed solution was extracted by vortex or oscillation for 10 min, centrifuged for 5 min, and the upper phase was collected. n-Hexane of 10 mL was added into the lower phase, and the above extraction and centrifugation operations were repeated once. The upper phase was combined, and evaporated to near dryness by rotating at 40 ℃, and the residual solution was blown to dryness by nitrogen. The residue was dissolved and diluted to 5 mL by methanol, and the resulting solution was passed through a 0.22 μm filter membrane. The filtrate was introduced into the ultra-high performance liquid chromatograph-triple quadrupole mass spectrometer. Seven lipid-soluble vitamin nutritional supplements were separated on HSS T3 column (100 mm×2.1 mm, 1.8 μm) by gradient elution with mixed solutions composed of methanol solution containing 0.1% (volume fraction, the same below) formic acid and 0.1% formic acid solution at different volume ratios, ionized by ESI⁺ mode, detected by MRM mode, and quantified by matrix matching method. It was shown that linear relationships between values of the mass concentration and the peak area were kept in the ranges of 50-1 000 μg·L^-1 (vitamin A acetate, vitamin A palmitate, DL-α-vitamin E acetate) and 10-200 μg·L^-1(vitamins D₂, D₃, K₁, K₂), with detection limits (3S/N) in the range of 2.06-7.56 μg·L^-1. Test for recovery was made according to standard addition method, giving recoveries in the range of 83.4%-106%, and RSDs (n=6) of the determined values ranged from 1.9% to 4.9%. The proposed method was applied to the analysis of actual samples, and the detection amount and label value of each lipid-soluble vitamin nutritional supplement were basically consistent.