P-glycoprotein (P-gp)/MDR-1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P-gp inhibitors were reported to reverse the MDR when co-administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P-gp inhibitors tested in the clinics are reported to have cross-reactivity with CYP450 drug-metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co-administered chemotherapeutic drugs. In this study, two piperine analogs (3 and 4) having lower cross-reactivity with CYP3A4 drug-metabolizing enzyme are identified as P-glycoprotein (P-gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over-expressing P-gp (KB Ch R 8-5, SW480-VCR, and HCT-15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB Ch R 8-5 cells and found to act as competitive inhibitor of P-gp as well as potentiated the vincristine-induced NF-KBmediated apoptosis. Therefore, 3 ((2E,4E)-1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-5-(4-hydroxy-3-methoxyphenyl)penta-2,4-dien-1-one) can serve as a potential P-gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer. 52 | SYED Et al. 1 | INTRODUCTION P-glycoprotein (P-gp/MDR-1) belonging to the ABC family of efflux pump transporters plays an important role in the development of drug resistance in cancer (Ghaleb et al., 2018; Waghray & Zhang, 2018). From the clinical studies, it is evident that the expression of MDR-1 is inversely proportional to the efficacy of first-line chemotherapy (Chung, Santiago, De Jesus, Trinidad, & See, 2016). Moreover, some of the clinical studies have also revealed an increased expression of P-gp after chemotherapy, which positively correlates with the relapse of the tumor (Chevillard et al., 1996; Zhou,