OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the simultaneous determination of the concentrations of verapamil and norverapamil in human breast milk and apply it in clinical practice. METHODS Milk samples(100 μL) were precipitated with 300 μL of methanol containing internal standard(5 ng·mL^-1 verapamil-d₃), and the supernatant was taken for analysis after vortexing and centrifugation. The chromatographic separation was performed on a Waters ACQUITY BEH C₁₈ column with gradient elution at 0.4 mL·min^-1, where mobile phases A and B were 0.1% formic acid-10 mmol·L^-1 ammonium acetate-water and methanol, respectively. The injection volume was 2 μL, and the analysis time was 4 min. The detection of the analytes was performed by electrospray ionization in positive mode by multiple reaction monitoring with the transition of m/z 455.3→165.2(verapamil), m/z 441.5→165.3(norverapamil) and m/z 458.3→165.0(verapamil-d₃). The established LC-MS/MS method was validated, and the method was used to determine the drug concentrations in breast milk of lactating patients. RESULTS The calibration curves for verapamil and norverapamil exhibited good linearity within the concentration range of 0.5 to 100 ng·mL^-1 (r=0.999). The intra-assay and inter-assay accuracies were both within ±15% of the labeled values, and the relative standard deviations were less than 15%. The extraction recovery, matrix effect, and stability all met the acceptance criteria for bioanalytical method validation. This analytical method was effectively employed to quantify drug concentrations in the breast milk of a lactating patient. The verapamil concentration in the breast milk of the patient ranged from 1.80 ng·mL^-1 to 10.73 ng·mL^-1, while norverapamil concentrations varied from 2.15 ng·mL^-1 to 5.15 ng·mL^-1. The relative infant dose of verapamil in this patient was approximately 0.15%. CONCLUSION The established method is simple, rapid, and sensitive. It is suitable for monitoring drug concentrations in clinical milk and can provide reference for safe drug use during lactation.