Serpins are a family of protease inhibitors that use a complex mechanism of conformational change in order to inhibit target proteases. Although the structural mechanism of serpin function is well characterised, the biological roles and targets of many serpins remain elusive. Among the few characterised Drosophila serpins, Serpin4 is of particular interest as it encodes multiple isoforms that are predicted to have different sub-cellular locations and different target specificity. To investigate the roles of Serpin4 to help predict target proteases we over expressed Serpin4 transgenes in various Drosophila tissues. We found that over expressing Serpin4 isoform A in ovarian follicle cells causes a disruption in cross-linking of the innermost eggshell layer, the vitelline membrane. As Serpin4A inhibits proteases that cleave at Arg-Arg-Lys/Arg residues, we have identified the eggshell protease Nudel as a candidate target. Consistent with this idea, Nudel proteolysis is affected when Serpin4 is over expressed. Using these results, combined with Serpin4 localisation data, we propose a model for the interaction between Serpin4 and Nudel following its role in eggshell biogenesis. Future experiments will focus on confirming the interaction between Serpin4 and Nudel in vivo, leading to a greater understanding of the proteases involved in eggshell biogenesis and how they function to produce this complex structure.