In kinetoplastids, the first seven steps of glycolysis are compartmentalized into a glycosome along with parts of other metabolic pathways. This organelle shares a common ancestor with the better-understood eukaryotic peroxisome. Much of our understanding of the emergence, evolution, and maintenance of glycosomes is limited to explorations of the dixenous parasites, including the enzymatic contents of the organelle. Our objective was to determine the extent that we could leverage existing studies in model kinetoplastids to determine the composition of glycosomes in species lacking evidence of experimental localization. These include diverse monoxenous species and dixenous species with very different hosts. For many of these, genome or transcriptome sequences are available. Our approach initiated with a meta-analysis of existing studies to generate a subset of enzymes with highest evidence of glycosome localization. From this dataset we extracted the best possible glycosome signal peptide identification scheme for in silico identification of glycosomal proteins from any kinetoplastid species. Validation suggested that a high glycosome localization score from our algorithm would be indicative of a glycosomal protein. We found that while metabolic pathways were consistently represented across kinetoplastids, individual proteins within those pathways may not universally exhibit evidence of glycosome localization.