Advancements in analytical technologies have reshaped our understanding of the ribosome, revealing its potential as a regulatory player in gene expression beyond its canonical role in mRNA translation. Emerging evidence suggests that post-translational modifications (PTMs) of ribosomal proteins contribute to proteome diversity and translational regulation. Here, we present a novel two-dimensional size exclusion chromatography (2D-SEC) technique integrated with tandem mass spectrometry (MS) for the comprehensive analysis of ribosomal heterogeneity and PTMs. Using Saccharomyces cerevisiae as a model, we explored the role of ribosomal protein methylation by analysing ten methyltransferase knockouts, each targeting specific methylation sites. Our 2D-SEC method achieved high-resolution separation of ribosomes from whole-cell lysates into polysome, monosome, and subunit populations, enabling targeted purification and in-depth PTM characterization. Quantitative MS revealed dynamic changes in ribosomal protein methylation across translationally active (polysome) and inactive (unassembled subunit) ribosome populations. Notably, methylation state variations were detected at six of twelve known methylation sites on yeast ribosomal proteins. This workflow offers a robust and reproducible approach to studying ribosome heterogeneity, significantly improving the resolution and accessibility of PTM analysis. Furthermore, the technique is adaptable to investigating ribosome-nascent chain complexes and cellular responses to environmental stimuli, expanding its utility in understanding ribosomal regulation. By integrating advanced chromatographic and mass spectrometric techniques, this study underscores the transformative potential of new technologies in decoding ribosome dynamics and PTM-mediated regulation.