The determination of post-translational modifications is one of the main challenges in proteomics research. Mass spectrometry is a powerful tool for the structural characterization of proteins and different mass spectrometric techniques for the analysis of post-translational modifications of individual proteins or protein populations have been developed. This review describes the most recent advances in mass spectrometry-based approaches for the detection and determination of post-translational modifications, with an emphasis on glycosylation and phosphorylation.