A new series of 5-acetylbarbituric based thiosemicarbazones has been synthesized from 5-acetylbarbituric acid and unsubstituted/substituted thiosemicarbazides (1-6). The synthesized compounds were well characterized by elemental analyses, FT-IR, ¹H, ¹³C NMR and mass spectroscopic methods. Three dimensional molecular structure of three compounds (1, 2 and 6) was determined by single crystal X-ray crystallography. The compounds were evaluated for their in vitro cytotoxicity against HeLa-229 cancer cell line.