3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the universal donor of sulfate to sulfotransferases, a large group of enzymes that transfer sulfate to various molecules in the sulfation pathway. Methods that have been previously used to determine this important metabolite lack in specificity. The aim of the project was to develop a method for determining PAPS in cells and cellular fractions, using hydrophilic interaction liquid chromatography and mass spectrometry. The developed method gave a retention time under 10 minutes, acceptable chromatographic efficiency and satisfactory repeatability in measurements. The method was able to separate PAPS from ATP and ADP, which could interfere with PAPS signal in the MS as they share mass spectrometric features. In addition, a simple sample preparation procedure was developed using ultra centrifugal filters. The method was evaluated regarding linearity, carry-over, within- and between-day precision etc., and PAPS levels was estimated in MDCK cell lines and their Golgi fractions, also following treatment with sodium chlorate (a PAPS synthesis inhibitor). The combination of the ultra centrifugal filtration, as a sample preparation step, ZIC-pHILIC and ESI-MS detection was found to be a suitable technique for the analysis of PAPS.