Objective To develop an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining lutein and natural vitamin K (VK) homologues, including phylloquinone (VK₁), menaquinone-4 (MK-4), menaquinone-7 (MK-7), and menaquinone-9 (MK-9), in poultry eggs by optimizing ultrasound assisted enzymatic hydrolysis.Methods The target compound is extracted from poultry egg samples by ultrasound assisted enzymatic hydrolysis and then separated on C₁₈ (100 mm×2.1 mm, 3.0 μm) column with 0.1% formic acid aqueous solution-0.1% formic acid methanol for gradient elution. Meanwhile, the samples are quantified by external standard with APCI source for detection and analysis.Results Under the ultrasound assisted enzymatic hydrolysis conditions of enzyme dosage of 1 500 U and hydrolysis time of 1 hour, the linear relationships of lutein and natural VK homologues are good in the range from 1.0 ng/mL to 5 000 ng/mL (R²>0.996). The detection limit is 0.8~1.5 μg/100 g, and the quantification limit is 2.0~5.0 μg/100 g. The average recovery of lutein and natural VK homologues is 80.1%~94.9% when the spiked concentration is 10, 20, and 100 μg/100 g with the RSD of 1.3%~5.5%.Conclusion This method has high detection efficiency, sensitivity, and selectivity and can be used as a detection method for lutein and natural VK homologues in poultry eggs.