Updated project metadata. Quantitative high-throughput screening (qHTS) pharmacologically evaluates libraries of drugs and investigational agents for potential therapeutic uses, toxicological risk assessment, and increasingly for academic chemical tool discovery. Phenotypic HTS assays aim to interrogate molecular pathways and networks, often relying on cell culture systems, historically with less emphasis on multicellular organisms. C. elegans has served as a powerful eukaryotic model organism for human biology and disease by virtue of genetic conservation and experimental tractability. Here we describe a paradigm to enable C. elegans in qHTS using 384-well microtiter plate laser scanning cytometry for rapid signal acquisition with concurrent quantification of the fluorescent protein-expressing organism to select phenotype-modifying compounds for subsequent life-stage and proteomic analysis. E. coli bacterial ghosts, a non-replicating nutrient, allow compound exposures over 7-days spanning two life cycles to mitigate complications from bacterial overgrowth. We demonstrate the method using 643 anti-infective biased agents tested in 7-pt titration to assess feasibility of nematode-based in vivo qHTS. A pharmacological profile from the primary screen confirmed the efficacy of known anti-parasitic molecules, such as ivermectin and levamisole as well as illuminating anthelmintic properties of general chemical classes, including MEK, -secretase, bromodomain and proteasome inhibitors. Subsequent life-stage and proteomics analysis helped elucidate the physiologic consequence of specific compounds on C. elegans viability. For example, bortezomib induced a parallel upregulation of core proteasome subunits and the transcription factor skn-1-activating DDI aspartyl protease, while the kinase inhibitor trametinib upregulated the C. elegans MEK-1 ortholog, Mnk-1, downstream aminoacyl tRNA synthetases and ribosomal survival pathway proteins. We anticipate a broader application of this qHTS-coupled proteomics approach will enable the analysis of C. elegans orthologous transgenic phenotypes of human pathologies to facilitate drug and probe profiling from high-impact chemical libraries for a range of therapeutic indications.