Abstract The escalating scarcity of traditional protein sources poses a critical challenge to the sustainable development of animal agriculture. However, in the process of exploring alternative protein resources, the potential resource of glutamate wastewater treatment solution (GWT) was discovered. GWT contains substantial amounts of crude protein, offering an innovative avenue to mitigate protein source shortages. Given its high crude protein content, to investigate this hypothesis, Drosophila melanogaster larvae were selected as a model organism. The effects of GWT on larval metabolic processes, after substituting GWT for yeast in the larval diet, were evaluated using metabolomic techniques. These techniques included principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), variable importance in the projection (VIP) analysis, and metabolite comparisons against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and Human Metabolome Database (HMDB). Metabolomic profiling revealed significant upregulation of amino acids, peptides, and their analogues in larvae fed with GWT. This finding was corroborated by biochemical assays that showed increased total protein content, indicating that larvae efficiently converted GWT into their endogenous proteins. In addition, the larvae contained the amino acids, peptides, and their analogues required for feed, highlighting their great potential as an insect protein feed source. In summary, our findings provide a theoretical foundation for developing novel protein resources and herald broad prospects for the application of GWT in the feed industry.