Despite the availability of various classes of chemotherapy agents for the treatment of the complicated disorder, cancer, developing most effective cytotoxic agents with high potency and least drawbacks being major concern in the field of medicinal chemistry. Therefore, the demand for novel molecules to treat cancer efficiently through multiple mechanisms is increasing. It is very much evident that bischalcones being the global research focus to compensate the demand. Fortunately, the development of the most appropriate bischalcone derivatives with high potency and binding affinity still not been addressed. Hence, emphasizing eco-friendly technological shift, in this research, ultrasonic technique was used to synthesis series of bischalcones derivatives, RVD1-RVD4. The potential cytotoxicity of the compounds was confirmed through in-vitro evaluation using Brine Shrimp (Artemia salina) Lethality Assay. Among the compounds tested, compound RVD3 and RVD4 has showed significant cytotoxicity at LD50 values 13.18 µg/mL ±0.12 and 13.80 µg/ml ±0.11 respectively. Consequently, in silico molecular docking studies have also been performed to evaluate the possible underlying mechanism of action of the compounds against Dihydrofolate Reductase enzyme (DHFR) anticancer drug target. Molecular docking results revealed that the highly potent bioactive bis-chalcone RVD3 is less selective towards inhibition of DHFR. Keywords: Cancer, bis-chalcones, cytotoxic agent, ultrasonic, in-vitro brine shrimp (Artemia salina) lethality assay, molecular docking, Dihydrofolate Reductase (DHFR)