Protein translation is intricately controlled within the eukaryotic cell. While previously thought to be controlled exclusively through translation factors, it has recently emerged that the ribosome itself is important for translational regulation. Ribosome composition is highly varied within cells, a phenomenon called ribosome heterogeneity, and this is known to regulate selective mRNA translation. However, the contribution of protein post-translational modifications (PTMs) to ribosome heterogeneity remains poorly understood. Here we have combined ribosome profiling through Ribo-Mega SEC (size exclusion chromatography) with mass spectrometry to systematically profile ribosomal PTMs in Saccharomyces cerevisiae and human K562 cells. Ribo-Mega SEC allowed separation of distinct pools of ribosomes, from translationally active polysomes to unincorporated subunits, for downstream mass spectrometric analysis. Through use of multiple different proteases for protein digestion, we identified every yeast and human ribosomal protein (RP) except RPL41, with peptides covering every single residue for the vast majority of RPs. This includes paralogous pairs of RPs that differ by only a few residues. We identified and quantified 12 methylation sites on yeast RPs and six methylation sites on human RPs, confirming that these are all present on actively translating ribosomes. In both yeast and human, half of the methylation sites were found to be substoichiometric on polysomes, indicating that these methylation sites contribute to ribosome heterogeneity. Through phosphopeptide enrichment, we identified over 100 phosphorylation sites on actively translating yeast ribosomes. Remarkably, most of these were found on the exterior of the ribosome, suggesting they may be actively regulated on intact ribosomes. We also identified several other PTMs on ribosomal proteins, including acetylation and ubiquitination. Together, our results reveal that PTMs contribute significantly to ribosome heterogeneity and provide a foundation for detailed studies on the roles of PTMs in translational regulation.