Worldwide, the vitamins and minerals are the most widespread dietary supplements used by populations. The validated UHPLC method was demonstrated to be precise, accurate, and robust for the simultaneous determination of vitamins D3 (CT and CHL) and K2 (MK-4 and MK-7) in dietary supplements. The validation results indicated that the method had reasonable linearity ( R 2 ≥ 0.9990), good recovery (>82%), satisfactory intra-day precision (≤1.9%) and inter-day precision (≤3.5%), and high selectivity and specificity. The limits of detection and limits of quantification for the target nutritional compounds ranged between 0.04–0.05 µg/mL, respectively. The overall runtime per sample was above 35 min, with the retention times of 2.40, 6.59, 7.06, and 32.6 min for vitamin D3 (CT and CHL) and vitamin K2 (MK-4 and MK-7), respectively. In this regard, the simultaneous determination of vitamin D3 (calcitriol-CT and cholecalciferol-CHL) and K2 (menaquinone-4-MK-4 and menaquinone-7-MK-7) in dietary supplements was developed by using ultra-high-pressure liquid chromatography (UHPLC). Accurate determination of vitamins is important for the improvement of dietary supplement quality and nutrition assessments. The content and composition of dietary supplements is of great interest due to their increasing consumption and variety of available brand offered in the market.
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