The determination of nucleotide profile in human milk is an important issue for making nucleotide-fortified dairy products. Here, a method for the simultaneous quantitation of five monophosphate nucleotides (cytidine 5′-monophosphate (CMP), uridine 5′-monophosphate (UMP), adenosine 5′-monophosphate (AMP), inosine 5′-monophosphate (IMP), guanosine 5′-monophosphate (GMP)) in milk using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed. The nucleotides were separated using a C18 column and gradient elution with water containing 0.1% (v/v) formic acid and acetonitrile. The method was validated with spike recoveries of 90.3–102.7% and 81.2–96.2%, and repeatability relative standard deviations of 2.5–4.3% and 2.6–4.6% for fresh bovine milk and milk powder, respectively. The method was applied for the assay of Chinese human milk samples collected from 59 nursing mothers with 2–4 months of lactation in Zhejiang, China. The mean (±SD) contents of CMP, UMP, AMP, IMP, and GMP are 3.1 ± 1.8, 2.2 ± 2.2, 0.23 ± 0.18, 0.018 ± 0.023, and 0.24 ± 0.19 mg.kg−1, respectively. The results of this work may serve as a guideline for the humanizing bovine milk-based infant formulas by supplementing the five monophosphate nucleotides for Chinese infants.