We have developed a protocol to isolate an excreted-secreted protein (ESP) fraction by collecting and concentrating the spent media from synchronized large-scale C. elegans cultures. Using high-throughput liquid chromatography/ mass spectrometry analysis, we discovered that the C. elegans ESP fraction contains C-type lectins domain-containing proteins, proteases, antimicrobial proteins and lysozymes. Many ESP protein components have been previously identified as transcriptionally upregulated upon pathogen exposure, which agrees with the innate immune response role assigned to some of these proteins. In addition, many uncharacterized proteins were found in this preparation, including nematode-specific proteins. Ongoing efforts include the analysis of the evolutionary rate of these excreted-secreted proteins. This study will contribute to our understanding of how pathogen recognition and defense mechanisms evolve and provide insight on proteins that could be key players in host-pathogen interactions.