The ribosome is one of life’s most important molecular machines. However, it has historically been seen as a backstage participant in gene regulation, binding to mRNA and translating codons in an unregulated fashion. However, recent evidence suggests that core protein components of the ribosome can be regulated and diversified as a means to intricately manipulate the expression of the cellular proteome. Post-translational modifications (PTMs) of ribosomal proteins are likely one such means of regulation. Here we have investigated the roles of methylation sites present in the Saccharomyces cerevisiae ribosome by use of ten methyltransferase knockouts, each of which adds one or two distinct methylation sites to the ribosome. These knockouts were utilized in a two-stage approach. The first stage involved employing polysome profiling experiments, a robust analytical technique for purifying ribosomes and assessing cellular translational health. In the second stage, we employed quantitative mass spectrometry, which provided valuable insights into the impact of methylation alterations on ribosome protein PTM dynamics and the global effects on ribosomal heterogeneity. Novel methods of purification and validation were developed to characterise the ribosome and its PTMs with a 2-dimensional size exclusion chromatography technique. This new method successfully separated ribosomes from whole cell lysate with resolution at the polysome, monosome, and subunit level. It also allowed the relative quantity of methylation in different ribosomal populations to be evaluated, for instance, actively translating polysomes versus subunits not assembled into complete ribosomes. Mass spectrometry results demonstrated variations in the methylation state at six of the twelve sites on the yeast ribosome, one such effect suggesting that the loss of methylation on the K4 site of RPL12 impacts the methylation state of the N-terminus, which may play a functional role in of translation. Our approach to the separation of ribosomes in yeast and downstream analysis of modifications provides a path to improve both robustness and ease of analysing ribosomal heterogeneity.