The improved pre-column methods followed by gradient elution C18-chromatography (C18-UFLC) with photodiode (DAD) and fluorescence (FLD) detection for analysis of tocotrienols (T3s), tocopherols (Ts), α-tocopheryl acetate and cholesterol in plant, algae and fish oils, milk and animal tissues have been described. C18-chromatography without saponification permitted quantification of T3s and Ts in oils. Quantification of tocols in milk involved saponification followed by C18-chromatography. β-tocol and γ-tocol were unseparated using C18-chromatography. Esterification of hydroxyl group of tocols with trifluoroacetic anhydride allows their satisfactory separation. The combination of esterification of tocols, C18-chromatography and DAD monitoring at 278 and 205 nm provide the suitable analytical tool for quantification of β- and γ-forms of tocols in biological samples. Our original C18-chromatographic methods are satisfactory precise, accurate, repeatable and offer low values of limit of detection (<10 ng/mL) and limit of quantification (<27 ng/mL) for assayed tocols and cholesterol.