Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signalling functions indicating an importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment while automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems‐level we here establish an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO2 and Fe(III)NTA-IMAC based enrichment, and the most advantageous, species specific starting amount for S. pyogenes, L. monocytogenes, and B. subtilis. Our data represents, to the best of our knowledge, the largest phosphoproteome identified by a single study for each of these bacteria. For higher sample amounts (> 250µg) we observed a superior performance of Fe(III)NTA cartridges, while more peptides were identified from smaller sample amounts using TiO2-based enrichment. Both cartridges largely enrich the same set of phosphopeptides suggesting no improvement of peptide yield from complementary use of both cartridges. Distribution of S/T/Y phosphorylation varied between bacterial strains with threonine being the main site of phosphorylation in S. pyogenes, while in B.subtilis and L. monocytogenes showed the highest percentage of phosphorylation at serine.