The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected siRNA, which showed 73.6% inhibition in replication of Influenza A/pdmH1N1 and 62.1% inhibition in replication of Influenza A/H3N2 at 48 h post infection on MDCK cell line. This study provides a basis for the development of siRNA which can be used as universal anti-IAV therapeutic agent.