Ascorbic acid or vitamin C is one of the most important and well-known vitamins essential for proper functioning of the human body and it is a known antioxidant. In this thesis, a method was developed for qualitative and quantitative determination of ascorbic acid (AA) and dehydroascorbic acid (DHA) as its oxidation product; both compounds are considered as vitamin C. For this purpose, ultra-high performance liquid chromatography coupled with triple quadrupole mass detector was used in negative mode, by recording specific transitions. One quantitative and one qualitative transition were optimized, respectively, 175 → 115 m/z, 175 → 87 m/z for AA and 173 → 143 m/z, 173 → 71 m/z for DHA. A 0.1% aqueous solution of acetic acid was used as the mobile phase, while separation was performed using a C18 chromatographic column at a temperature of 20°C and a flow rate of 0.7 mL/min. Mass spectrometry was used with electrospray ionization, ionizer temperature of 350°C and capillary voltage of -3600 V. The calibration curve was divided into two concentration ranges, from 0.5 µg/mL to 5.0 µg/mL and from 10 µg/mL to 50 µg/mL. The limits of detection and quantification were calculated from the signal-to-noise ratio and are respectively 2.1 ng/mL; 7.2 ng/mL for AA and 18.7 ng/mL; 62.3 ng/mL for DHA. By validating the accuracy, precision, and method robustness, as well as system suitability and stability of working standard solutions (which significantly decreases over 24 hours), the method's suitability for qualitative and quantitative determination of AA and DHA was confirmed.