Recently, new physiological roles of vitamin K homologues have been established in the treatment of rheumatoid arthritis, osteoporosis, hepatocellular carcinoma and leukemia. However, relatively high plasma protein binding, low plasma concentrations and occurrences of interfering lipids make accurate determination of vitamin K homologues a challenging task. Therefore, a sensitive and reliable salting-out assisted liquid/liquid extraction (SALLE) method coupled with HPLC-Fluorescence detection was designed for efficient extraction and quantification of trace levels of vitamin K homologues in human plasma. The investigated vitamin K homologues were phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4) and menaquinone-7 (MK-7). The method employed a new efficient fluorescence derivatization reaction using ethanolic solution of stannous chloride in acidic solution to generate highly fluorescent naphthohydroquinone derivatives. Correlation coefficients were more than 0.998 in the concentration ranges of 0.3–100ngmL−1 with detection limits of 0.1–0.17ngmL−1 in human plasma. The developed HPLC-FL system was successfully applied for sensitive determination of vitamin K homologues in plasma of healthy volunteers. The developed method may provide a valuable tool in the pharmacoinformatic studies concerning the roles of vitamin K homologues. Display omitted •A novel, highly sensitive and reliable SALLE method coupled with HPLC-FL method was developed for trace determination of vitamin K homologues in plasma.•SALLE method provided a simple and efficient extraction technique with better recoveries•Fluorescence derivatization using ethanolic solution of SnCl2 enhanced the method sensitivity and selectivity.•The proposed SALLE-HPLC-FL method was 2–500 times more sensitive as compared to the reported methods.•The developed method may provide a valuable tool for monitoring vitamin K homologues in different clinical studies.