Objective To establish an ion chromatography method for the simultaneous determination of six human milk oligosaccharides (HMOs) in infant formula powder.Methods The samples were enzymatically hydrolyzed using fructanase. Gradient elution was performed with a mobile phase consisting of water, 0.5 mol/L sodium hydroxide, and 0.3 mol/L sodium acetate. Separation was carried out on a Thermo Fisher CarboPac? PA1 Analytical Column, and detection was performed using ion chromatography with pulsed amperometric detection.Results In milk powder, 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), and lacto-N-tetraose (LNT) showed good linearity within the range of 1~100 mg/L, while lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL) exhibited good linearity in the range of 0.5~50.0 mg/L, with correlation coefficients all greater than 0.999. The limit of detection (LOD) for 2'-FL, LNnT, 3-FL, and LNT was 10 mg/100 g, and the limit of quantification (LOQ) was 30 mg/100 g. The LOD for 3'-SL and 6'-SL was 5 mg/100 g, and the LOQ was 15 mg/100 g. The average recoveries of the six HMOs at low, medium, and high concentrations ranged from 98.0% to 101.0%, with relative standard deviations (RSDs) between 0.3% and 2.4%. In actual sample analysis, when the content of the six HMOs ranged from 0.1 to 10.0 g/kg, the RSDs were 0.5% to 9.4%. When the content ranged from 10 to 100 g/kg, the RSDs were 1.4% to 4.0%.Conclusion The proposed method meets the requirements for routine analysis and is suitable for the determination of six HMOs in various infant formula powders containing HMOs.