The Non Structural 1 (NS1) is a 230–237 amino acid relatively well-conserved viral protein that is usually expressed only during infection [1]. It contains an RNA binding domain (RBD) and an effector domain (ED) that is connected by a variable linker [2]. The NS1 protein has been shown to be a key player that counters the host interferon (IFN) response to viral infection in strain-specific ways [3]. NS1 inhibits the function of OAS (2′–5′-oligoadenylate synthetase) and PKR (protein kinase R) [4] and block IFN-β synthesis at the post-transcriptional level by inhibiting the pre-mRNA splicing and blocking the export of poly(A)-RNAs from the nucleus to the cytoplasm [5]. It also hampers the host’s defense pathways by interfering with the host RNAi pathway, adaptive immune response, and the apoptotic response [6]. NS1 is also a key component in the temporal regulation of viral RNA synthesis, its splicing, and translation [7]. Thus, NS1 enhances the virulence of infection, posing as a compelling target for influenza treatment. These compounds will enable researchers to test how NS1 inhibition impacts the infection cycle and how antagonists can be leveraged alone or in combination with existing agents in the next generation of treatments for influenza infection. We developed and conducted a high-throughput screen using a novel yeast-based phenotypic assay to identify compounds which specifically inhibit NS1 function. Here we report a potent NS1 antagonist chemical series, represented by ML303. The SAR (structure activity relationship) around this chemical series was further developed by measuring the compound’s ability to inhibit replication of the influenza A/PR/8/34 virus in MDCK cells. The ML303 compound series specifically restored the NS1-inhibited expression of the interferon mRNA, inhibited the NS1 function in mechanistic assay and exhibited a potent antiviral activity in culture.