Phosphorylation is an essential regulatory protein modification, which modulates many presynaptic processes, including synaptic vesicle trafficking. Protein phosphorylation impacts neurotransmitter release and is likely to be mechanistically important for presynaptic plasticity, although only a few mechanisms have been described to date. Thus, interactomics and phosphoproteomics are important tools to determine presynaptic mechanisms. Over the last two decades, mass spectrometry-based proteomics has become the method of choice for protein-protein and protein phosphorylation analysis. This protocol is based on well-established procedures in our laboratory and will describe the application of affinity purification of presynaptic proteins via GST-fusion protein pull-downs for the study of presynaptic phospho-signalling. The pull-down is followed by on-bead proteolysis, chemical stable isotope labelling for precise quantification, enrichment of phosphorylated peptides using TiO2, and LC-MS/MS. The method outlines the software used for peptide/protein identification and quantification, as well as some of the bioinformatics tools that are available for putting the regulated phosphorylation sites into a functional context.