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A high-performance liquid chromatography method was developed for separation, detectionand quantification of all water-soluble vitamins excluding vitamin B9. The method was aimedto suit both ultraviolet detection and mass spectrometry. Separation was done using a C18column with two different mobile phases. Mobile phase A consisting of 0,05% formic acid andwater (pH 2,95) and mobile phase B which consisted of methanol with 0,05% formic acid (pH2,95). These mobile phases were used in a combined isocratic and gradient elution. Diode arraydetection was used at three different wavelengths (210, 257 and 270nm) for the ultravioletdetection, while a high-resolution quadrupole time of flight mass spectrometer was used forthe MS. Liquid-liquid extraction was used as a preparation of the plasma samples with n-heptane as the organic phase. The method aimed to be validated according to ICH guidelinesfor bioanalytical methods. The HPLC-UV method showed promising results with goodrepeatability and fulfilling the aim of separating and detecting the vitamins faster than existingmethods. However, the sample preparation did not fulfill the requirements and neithervalidation nor quantification could be performed with this method. The HPLC-MS methodshowed more promising results as it could be validated and used to quantify samples albeit therepeatability and accuracy were not good enough. This is mainly due to the absence of aninternal standard in the analysis. These methods also fulfilled the aims of separating anddetecting the vitamins faster than existing methods.