Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post-translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two-dimensional gels after differential phosphoprotein staining or gel-free by stable isotope labeling, sequential phosphopeptide enrichment and following LC-MS analysis. In the current work, we combined in a proof-of-principle experiment these techniques using N-14/N-15 metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC-MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide-based gel-free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.