INTRODUCTION: Although there are indications that insect-based proteins may have potential biomedical applications (anticancer and antimicrobial), as well as in cellular agriculture (food and feed), they have not been sufficiently investigated. The hemolymph of insect larvae is protein-rich, particularly in storage proteins that are involved in amino acid metabolism and protein synthesis. In order to characterize these proteins, the first step is their successful isolation. Using diapausing 5th instar larvae of the economically important European corn borer moth (ECB) Ostrinia nubilalis (Hbn.) as a model system, in this study we optimized a method for isolating individual native hemolymph proteins from polyacrylamide gels and we performed initial tests of isolated proteins bioactivity. OBJECTIVES: The main objective in this study was to optimize an easy and affordable method for isolation of individual hemolymph proteins in the native state, without the use of chemicals that would affect their structure and function (e.g. sodium dodecyl sulfate, SDS). This allows further testing of these proteins for biomedical and application in cellular agriculture, and further work with isolated proteins in downstream in vitro proteome research, which will bring new knowledge and directions for different in silico proteome research. METHOD / DESIGN: Hemolymph was collected from diapausing 5th instar ECB larvae, after which hemocytes were removed from the hemolymph by centrifuging the samples for 30 min. at 16 000 g. Hemolymph proteins were separated by native polyacrylamide gel electrophoresis (PAGE) on a customized discontinuous gel without a well comb, using the BIO-RAD Mini-PROTEAN® Tetra cell. In order to determine the position of protein fractions of interest on the gel after electrophoresis, thin vertical strips were cut from the sides of the polyacrylamide gel and stained with Coomassie Brilliant Blue, after which the same gel strips were destained. The strips were placed next to the original gels and 5 protein fractions were cut from the unstained part of the polyacrylamide gel, chopped and transferred to microtubes. Ultrapure water was added to the tubes and they were placed on the Biometra TSC ThermoShaker overnight at 30°C to elute the proteins from the gels. After elution, the protein samples were centrifuged for 15 min. at 10 000 g. The concentration of isolated proteins was determined by measuring the absorbance at 230 nm using the Shimadzu BioSpec-nano, with a serial dilution of bovine γ-globulin used as the protein standard. To confirm that the proteins were well isolated, the individual fractions were run in duplicate wells on discontinuous native PAGE using the BIO-RAD Mini-PROTEAN® 3 Cell, after which the gels were stained, destained and imaged. Finally, the effect of successfully isolated proteins on MRC-5 cell viability was examined using an MTT assay. RESULTS: Five distinct protein fractions were detected after the first native PAGE (P1-P5). After elution from the gel, these fractions and the method for their isolation were validated with a second native PAGE. Regarding the testing of isolated protein bioactivity, the results of the MTT assay indicate an antiproliferative effect of all 5 protein fractions, especially in the P4 fraction. CONCLUSIONS: The insect hemolymph protein extraction method optimized in this study proved to be simple and successful and could potentially be applied to other insect species as well. Also, the structure and function of the proteins remained intact during the isolation process, which allows further use of the isolated proteins in downstream in vitro proteome research, the results of which will contribute to protein identification and in silico proteome research based on different bioinformatics tools (e.g. protein-protein interaction analysis, in silico bioactivity analyses, etc.). Finally, since the isolated proteins showed antiproliferative effects on the selected cell line, their anticancer and antimicrobial activity will be further tested.