Abstract: Nucleotides are biologically important small molecules composed of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose) and at least one phosphate group. They play important role in improving gastrointestinal tract repair after damage, maintaining the immune system and regulating lipoprotein metabolism. Nowadays the role of dietary nucleotides has been paid increased attention in infant nutrition. Dietary nucleotides are crucial to maintaining normal growth and development in infants. Therefore the contents of nucleotides in infant milk powder become important. A method for the determination of five nucleotides (cytidine 5’-monophosphate (CMP), guanosine 5’-monophosphate (GMP), uridine 5’-monophosphate (UMP), adenosine 5’-monophosphate (AMP) and inosine 5’-monophosphate (IMP)) in infant milk powder by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The nucleotides in sample were hydrolyzed by alkaline phosphatase to the corresponding nucleosides. The nucleosides were adsorbed by selective boric acid solid phase extraction. After purification and elution, the chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (2.1 mm×100 mm×1.8 μm), and gradient eluted by 5 mmol/L ammonium formate and acetonitrile, and analyzed by HPLC-MS/MS in multiple reaction monitoring (MRM) mode via positive electrospray ionization. The contents of nucleotides were quantitatively analyzed by internal standard method. The results showed that the linear ranges of five nucleotides in infant formula are good, the correlation coefficients are above 0.998. Five kinds of nucleosides can be separated within 8 min. Cytidine, inosine and guanosine are all linear in the concentration range of 5-200 μg/L, adenosine and uridine are linear in the concentration range of 1.40 μg/L and 25-1 000 μg/L, respectively. The recoveries are between 74.2% and 110.7% with the relative standard deviations from 0.8% to 5.3% (n=6). The limits of quantitation of CMP, AMP, GMP, UMP and IMP are 0.7, 0.1, 0.7, 3.3, 0.6 mg/100 g, respectively. In order to study the accuracy of the method, 3 infant milk powder were analyzed. The method is sensitive, accurate, has good reproducibility and is suitable for the simultaneous determination of nucleotide content in infant formula products.