This study focuses on developing and evaluating a novel series of sulfapyridine derivatives for their potential as anticancer agents. The strategic starting material, 2-chloro-N-(4-(N-(pyridine-2-yl)sulfamoyl)phenyl)acetamide, served as the foundation for the synthesis of various compounds. Clarifying the synthetic compounds' structures was accomplished through the analysis of IR, ¹H NMR, ¹³C NMR, and MS spectral data. In vitro screening assays were conducted to assess the freshly generated compounds' anticancer activity in opposition to a panel of human cancer cell lines, which included (HePG-2), (PC-3), (HCT-116), and breast cancer cell lines from the mammary gland (MCF-7). Interestingly, compounds with a thiophene moiety compounds 4 and 8 in particular showed notable activity. Compound 8 demonstrated encouraging results against each of the four cancer cell lines, suggesting its potential as a lead candidate for further expansion as an anti-tumor agent. This study contributes to understanding the structure-activity relationship of sulfapyridine derivatives and highlights the potential of compound 8 as a valuable applicant for future anticancer drug development.