A new series of di-spirooxindole analogs, engrafted with oxindole and cyclohexanone moieties, were synthesized. Initially, azomethine ylides were generated via reaction of the substituted isatins (isatin, , 6-chloroisatin, , 5-fluoroisatin, , 5-nitroisatin, , 5-methoxyisatin, , and 5-methylisatin, , and (2)-octahydro-1-indole-2-carboxylic acid , in situ azomethine ylides reacted with the cyclohexanone based-chalcone to afford the target di-spirooxindole compounds . This one-pot method provided diverse structurally complex molecules, with biologically relevant spirocycles in a good yields. All synthesized di-spirooxindole analogs, engrafted with oxindole and cyclohexanone moieties, were evaluated for their anticancer activity against four cancer cell lines, including prostate PC3, cervical HeLa, and breast (MCF-7, and MDA-MB231) cancer cell lines. The cytotoxicity of these di-spirooxindole analogs was also examined against human fibroblast BJ cell lines, and they appeared to be non-cytotoxic. Compound was identified as the most active member of this series against prostate cancer cell line PC3 (IC = 3.7 ± 1.0 µM). The cyclohexanone engrafted di-spirooxindole analogs and (IC = 7.1 ± 0.2, and 7.2 ± 0.5 µM, respectively) were active against HeLa cancer cells, whereas NO substituted isatin ring and -fluoro-substituted (2,6)-2,6-dibenzylidenecyclohexanone containing (IC = 7.63 ± 0.08 µM) appeared to be a promising agent against the triple negative breast cancer MDA-MB231 cell line. To explore the plausible mechanism of anticancer activity of di-spirooxindole analogs, molecular docking studies were investigated which suggested that spirooxindole analogs potentially inhibit the activity of MDM2.