Vitamin K1 is a fat-soluble vitamin essential for human health. We used the Association of Official Analytical Chemists(AOAC) method 999.15, validated by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use(ICH) Guideline, to monitor vitamin K1 content in infant formula in the Republic of Korea. The Certified Reference Materials (CRM), NIST 1849a, were used for validation of the test method. The parameters of validation were linearity, range, specificity, accuracy, precision, LOD, LOQ and recovery. The linearity, R2 was above 0.999 and the analytical range was 0.001 to 0.22 μg/mL. The specificity was confirmed by the retention time (RT) and positive/negative sample test. The accuracy, precision and recovery values were 101.13 ± 0.13 %, 3.15 % and 81.60 % - 99.78 %. The LOD and LOQ values were 0.582 ng/mL and 1.76 ng/mL, respectively. Five livestock products inspection agencies voluntarily participated in the collaborative study. The HORRAT value was 0.6, which was within the reference value 0.5 – 2.0. The validated test method was applied for the monitoring of vitamin K1 in infant formula in the Republic of Korea. The results also conformed to 'The Standard for Livestock Products of Processing and Ingredient Specifications' and 'The Standard for Livestock Products Labeling'.