Entomopathogenic nematodes (EPNs) are obligate insect parasites commonly used in the biocontrol of many insect pests. They produce bioactive molecules referred to as excreted/secreted products (ESPs). The ESPs can interfere with the host immune system, increasing infection efficiency and chances of EPNs survival and reproduction. Therefore, understanding the composition and function of ESPs is vital for unraveling the mechanisms underlying nematode pathogenicity and improving their efficient usage as biocontrol agents. Our aim was to assess the effect of various activation materials on the ESPs protein spectrum and to identify infection-specific molecules by comparison with the ESPs profiles of infective juveniles treated with water or phosphate buffer. Three activation materials prepared from larvae of Galleria mellonella, and two controls of water and phosphate buffer were used for the in vitro activation of Heterorhabditis bacteriophora infective juveniles. The produced ESPs were collected and subjected to LC-MS/MS analysis using RSLCNano system on-line connected to Impact II Ultra-High Resolution Qq-Time-Of-Flight mass spectrometer. Protein searches were performed against a protein database derived from H. bacteriophora reference genome. LC-MS/MS results revealed significant variations in the composition of ESPs following activation with different materials. We identified several proteins, e.g. ShTK domain proteins, fatty acid- and retinoid- binding protein, recently associated with pathogenicity and immune modulation. The activation materials influenced the abundance and diversity of ESP components, suggesting that these materials may play a crucial role in modulating the nematode's virulence. This study was supported by grant GAČR 23-06457S.