Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.