In this study, a series of novel N-alkyl phosphoramidate monoesters of 3’-azido-3’-deoxythymidine (AZT) were synthesized using two methods. The synthesized phosphoramidates 7a–e were evaluated for their cytotoxic activity in three human cancer cell lines (cervical cancer (HeLa), nasopharyngeal cancer (KB), and breast cancer (MCF-7)) and a normal dermal fibroblast cell line (HDF) using sulforhodamine B assay. Among the synthesized phosphoramidates, the highest cytotoxic activity was demonstrated by phosphoramidate 7d with the N-n-propyl substituent in all the examined cancer cell lines, and its activity was found to be about two fold higher than that of the parent nucleoside (AZT). Phosphoramidate 7d showed not only a high cytotoxic activity against cancer cell lines but also a low toxicity against normal fibroblast cells; its selectivity index was >3 for all the investigated cancer cell lines. A slightly lower cytotoxic activity was shown by phosphoramidates 7a, 7b, and 7e, whereas phosphoramidate 7c with the N-(2,2,2-trifluoroethyl) substituent exhibited the least cytotoxic activity in all the cell lines used.