Objective To develop a method for the simultaneous determination of lutein, zeaxanthin, α-carotene, and β-carotene in infant formula milk powder and natural foods by online solid phase extraction-liquid chromatography (online SPE-LC). Methods Samples were saponified with 50% potassium hydroxide solution at 70 ℃ for 15 min. Saponified solution was diluted with 70% ethanol, and after high-speed centrifugation, the supernatant was directly analyzed. A styrene-divinylbenzene copolymer-based SelectCore PSS-packed chromatographic column was chosen as the online SPE column, using 80% methanol-water as the initial loading solvent and acetonitrile/methyl tert-butyl ether as the washing solvent. A ChromCore C₃₀ column (4.6 mm×250 mm, 3 μm) was used as the analytical column, with acetonitrile-methanol (26:74, V:V, 0.4 g/L of ascorbic acid) and methyl tert-butyl ether as the mobile phase for gradient elution. The flow rate was 1.0 mL/min, and the detection wavelength was 450 nm. Results The 4 target analytes in infant formula milk powder and natural foods were well separated. Linear correlation coefficients for all target analytes were greater than 0.9999. The limit of quantification of method for lutein, zeaxanthin, α-carotene, and β-carotene were 0.77, 0.72, 0.94, and 1.40 μg/100 g, respectively. Average recovery rates of infant formula milk powder were 99.76%, 103.51%, 99.16%, and 97.92%, respectively. The relative standard deviations of method repeatability for all the analytes were less than 2.5%. Conclusion This method is accurate, reliable, and reproducible and can meet the quantitative requirements of lutein, zeaxanthin, and α-carotene, β-carotene in foodstuffs. It can be applied to the determination of real samples.