Objective To establish a method for simultaneous determination of nicotinamide mononucleotide (NMN) α, β isomers and nicotinamide adenine dinucleotide (NAD+) in foods by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry. Methods The samples were extracted by 5% methanol aqueous solution, and purified by HLB solid-phase extraction column and mixed mode anion exchange solid phase extraction column. The Waters ACQUITY UPLC?HSS T3 chromatographic column was used for the chromatographic separation of target analytes at 30℃. The gradient elution method was used and the flow rate was 0.2 mL/min. The mobile phase composed of 5 mmol/L ammonium acetate aqueous solution containing 0.1% (V/V) formic acid and methanol. Quantitative determination was performed at the multi reaction monitoring mode of mass spectrometer. The quantitative ion pairs of NMN and NAD+ were m/z 335.0/123.0 and m/z 662.0/540.0, respectively. Results Under the above conditions, the separation degree of α-NMN and β-NMN isomers was 5.56. The method showed a good linear relationship between peak area and concentration over the range from 10 ng/mL to 1000 ng/mL and the correlation coefficients of α-NMN, β-NMN and NAD+ were 0.9999, 0.9998 and 0.9995, respectively. The limits of detection of α-NMN, β-NMN and NAD+ were 4.0, 2.0 and 1.0 ng/mL and the limits of quantitation were 10.0, 5.0 and 3.0 ng/mL. The recoveries of α-NMN, β-NMN and NAD+ ranged from 93.8%?103.8%, and the relative standard deviations of precision were 2.1%?6.5%. Conclusion This method has the advantages of high sensitivity, good selectivity, accurate and reliable results, which is suitable for the simultaneous quantitative analysis of NMN α, β isomers and NAD+ in various matrix foods.