Abstract: This study was carried out to identify the synthesis of a new group of thiazole-linked thioureas with aromatic and aliphatic side chains (4a–k) using a one-pot three-component strategy which are well known for their material and medicinal properties. Using all the standard protocols, the synthesized compounds were assessed for alkaline phosphatase (AP) assay. 1H and 13C NMR were utilized for identification of the chemical structures of all the synthesized compounds. The density functional theory (DFT) studies and molecular docking studies of synthesized derivatives were carried out to understand the reactivity profile and binding behavior of all the compounds. The outcome of this study suggests that the synthesized compounds are potent alkaline phosphatase inhibitors whereas 4c and 4g are best compounds with lowest IC50 value i.e., 0.057 and 0.019 µM, respectively. The docking energies of 4c and 4g are highest among all the compounds i.e., −32.18 and −30.09 kJ/mol, respectively. The results suggested that these derivatives can be utilized in future for the synthesis of more potent inhibitors of alkaline phosphatase that can be used in future for treating different types of cancer especially breast cancer.