During cell differentiation, gene expression is regulated at multiple levels which is only partially captured by transcription profiling. In recent years it became increasingly clear that post-translational modifications of core histones and post-transcriptional regulation by RNA-binding proteins and microRNAs play an important role during differentiation. Recent advances in mass spectrometry-based quantitative proteomics now allow for genome-wide analyses at the protein level. This technology provides a powerful toolbox that can be used to study different levels of gene regulation and reveal their importance during development of multi-cellular organisms. We highlight recent studies and indicate how quantitative proteomics can be employed to investigate cell differentiation in the future.