Objective To establish an analytical mehod for the determination of nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD⁺) in health food by high performance liquid chromatography. Methods Determination was performed on Waters BEH-Amide column with mobile phase consisting of 0.1% phosphoric acid solution-acetonitrile (gradient elution). The flow rate was 1.0 mL/min, and the sample size was 2 μL. The column temperature was 35 ℃, and the detection wavelength was set at 263 nm. Health food was extracted with 60% methanol solution in ultrasonic water bath for 10 min. The extract was filtered through micoron filter, and analyzed by high performance liquid chromatography. Results The results showed that NMN and NAD⁺ exhibited excellent linear relationships, the correlation coefficient (r) was over 0.999 in the range of 10-1000 μg/mL. The method limit of quantitation was 0.50 g/kg. Recoveries were ranged from 95.9% to 98.8%, and relative standard deviations were ranged from 1.19% to 3.05%. Conclusion This method is accurate and suitable to determine NMN and NAD⁺ in health food.