Entomopathogenic nematodes produce bioactive molecules referred to as excreted/secreted products (ESPs). The ESPs comprise a mixture of proteins, lipids, glycans, and nucleic acids that can interfere with the host immune system in order to increase the chance of entomopathogenic nematodes to reproduce. Infective juveniles (IJs), the only free-living stage of nematodes, release ESPs typically in the early phase of infection when contacted with host tissues. The spectrum of ESPs has not yet been fully described, however, its quantitative and qualitative changes during the process of nematode infection were observed. Of particular importance are the immune-modulating proteins, such as proteolytic enzymes and their inhibitors, which have not yet been largely investigated. There are several approaches how to in vitro stimulate IJs to produce ESPs. We investigated the possible effect of various induction materials and time-related parameters on the yield of ESPs. We induced IJs of Heterorhabditis bacteriophora using four differently prepared Galleria mellonella homogenates and tested two time points of IJs induction and subsequent ESPs production. Based on our findings, the time of the IJs induction does not significantly affect the protein concentration of collected ESPs. However, we observed that the induction material affects the ESPs protein quantity. The collected ESPs were further characterised by mass spectrometry to identify proteins present in ESPs and suggest their function. According to GO annotation of mass spectrometric data, lipid-binding proteins, peptidase inhibitors, peptidases, and proteins with chitinase activity are among the most abundant groups of ESP components. As one of the major fractions determined by mass spectrometry were peptidases, we further focussed on their proteolytic activity and observed the presence of serine proteases. Our preliminary mass spectrometry data allow us to continue with identification and functional characterization of candidate bioactive molecules.