Gemcitabine, 2', 2'-difluoro-2'-deoxycytidine, pyrimidine antimetabolite, is an antineoplastic drug used to treat many advanced or metastatic cancers as monotherapy or in combination with other cytostatics, depending on the indication. The aim of this study was to develop a new, fast, simple and environmentally friendly capillary electrophoretic method for simultaneous identification and determination of gemcitabine and its impurity cytosine. Capillary electrophoresis was chosen as a suitable "green" alternative to an already developed HPLC method for the determination of gemcitabine and its impurities included in the 8th edition of European Pharmacopoeia. The analysis was carried out in 35 cm (27 cm) capillary at a temperature of 25 ° C and detection was at 275 i 237 nm. Three different buffers were studied while developing the method: borate (pH 9.3), phosphate (pH 7.0) and acetate (pH 3.0). Based on the experimental results, best buffer was shown to be the phosphate buffer (pH 7.0), while a better separation and peak shape were achieved by adding a higher concentration of SDS. 50 mM SDS was chosen as the optimal concentration. The effect of voltage on the analysis time and resolution between the analytes was investigated. It has been shown that a better separation of gemcitabine and cytosine was achieved at 10 kV. However, applying a lower voltage is prolonging the migration time of the analytes through the capillary. Therefore, it is necessary to take into account that a good resolution is achieved but that the analysis time is not too long. Further method optimization is required to achieve the appropriate peak separation, shape and symmetry with as short analysis time as possible.