COVID-19 is a disease caused by the highly transmissible and pathogenic SARS-CoV-2 virus. Since its first case was documented in 2019, it has rapidly widespread and has caused millions of deaths worldwide. Many intervention strategies targeting these proteins have been developed. However, frequently mutation of SARS-CoV-2 poses a challenge to the effectiveness of current treatments. Therefore, it is critical to develop new therapeutic drugs against this disease. In this present study, in silico approach was used to study the interaction between RetroMAD1™and SARS-CoV-2 proteins including Spike proteins (S), 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). The interaction of these viral proteins and RetroMAD1™ was performed through HDOCK server and visualised using PyMOL. Docking results revealed that all the complexes of SARS-CoV-2 proteins binding with RetroMAD1™ have relatively high docking scores. The binding energy of RetroMAD1™ complexes with SARS-CoV-2 S, 3CLpro, PLpro were − 15, -12.3 and − 15.4, respectively. RetroMAD1™antiviral efficiency and cytotoxicity was also evaluated using EpiAirway™ Model. In vitro validation of viral inhibitory effect of RetroMAD1™was performed with 3CLpro Inhibition Assay. The outcome showed that RetroMAD1™ represents a potential drug candidate against SARS-CoV-2 for its promising viral inhibitory effect.