A simple, reliable, and rapid hydrolysis method for quantitative determination of lutein and zeaxanthin in marigold and poultry feed was developed and analyzed by UPLC. Faster analysis time and better resolution were obtained by analysis columns with 1.7-µm particle materials in UPLC. The mean recovery for marigold was 85.4%–100% and for poultry feed was 83.4%–106.3%, relative standard deviation (RSD) was 8.75% or less. The concentrations of lutein and zeaxanthin in 20 marigold samples were assessed by the UPLC method and the HPLC method. The regression coefficients between concentrations of lutein obtained under the two methods were close to 1, and the regression coefficients of zeaxanthin were 0.96. Limit of detection (LOD) and limit of quantification (LOQ) of lutein in the two methods were the same values of 0.02 mg/100 g and 0.1 mg/100 g, whereas LOD and LOQ for zeaxanthin in UPLC were twofold lower than values of 0.04 mg/100 g and 0.2 mg/100 g in HPLC.