Background: Spodoptera litura is a significant pest of crops, causing substantial yield and economic losses. Overreliance on chemical insecticides has led to negative impacts on human health and the environment, as well as the emergence of pest resistance. As a sustainable alternative, Nuclear Polyhedrosis Virus (NPV) has been widely recognized as an effective biological control agent. This study aimed to explore the occurrence of S. litura NPV (Sl-NPV) across multiple regions in East Java Province, Indonesia. Methodology: Field surveys were conducted on maize and tobacco plants in 11 regions, including Mojokerto City, Mojokerto Regency, Jombang, Sidoarjo, Batu City, Malang, Lamongan, Kediri, Blitar, Pamekasan, and Bangkalan. Pathogenicity was evaluated using Koch’s postulate through spray applications on second-instar S. litura larvae. This study employed isolation of NPV from naturally infected S. litura larvae showing typical NPV infection symptoms. Findings: The exploration successfully identified Sl-NPV in Pamekasan and Blitar. Pathogenicity tests revealed that the isolate from Pamekasan was capable of inducing mortality in healthy larvae, whereas the isolate from Blitar did not produce infection symptoms when applied. These results highlight variability in pathogenic potential among isolates. Contribution: The findings provide important insights for the development of local Sl-NPV isolates as environmentally friendly biopesticides to support sustainable pest management in Indonesia.