Vitamin K homologues are highly lipophilic compounds that require long separation times on chromatographic analysis which does not meet the demand of higher sample throughputs in quality control laboratories. Therefore, this study aimed to develop a new validated high-throughput high-performance thin-layer chromatographic (HPTLC) method to quantify vitamin K homologues including phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4, vitamin K2), and menaquinone-7 (MK-7, vitamin K2). The densitometric analysis was carried out using HPTLC silica gel G 60 F254 plates as the stationary phase. The plates were developed with methanol-ethanol-isopropanol-water (75:5:5:15, v/v/v/v) in the absorbance mode at 254 nm. The retention factors of MK-4, PK, and MK-7 were 0.56, 0.43, and 0.23, respectively. Linearity was found to be in the range of 1-200 ng band1 for PK and MK-4 and 2-200 ng band1 for MK-7 with correlation coefficient of 0.9990 or more. The limits of detection and quantitation were 0.19-0.85 and 0.76-2.5 ng band1, respectively. The method was validated in accordance ICH guidelines. The method was applied for determination of vitamin K homologues in pharmaceutical formulations and food samples after extraction without prior clean-up procedures. The developed HPTLC method provides a useful tool for rapid and efficient high-throughput analysis of vitamin K homologues.