Histidine (His)-phosphorylation is labile at low pH and has therefore not been in the focus of proteomic analysis in the past although a few single-case studies have been performed. The systematic investigation of model substances generates confidence in experimental procedures and allows determining their limits. In order to extend earlier peptide studies to His-phosphoproteins and elucidate their behavior and recovery in proteomic procedures, potassium phosphoramidate (PPA) was used to generate model proteins, which were subsequently exposed to gel electrophoresis, enzymatic digest and mass spectrometry based protein analysis. Myoglobin having eleven His-residues was highly phosphorylated by PPA showing a distribution of modified protein forms with four phosphate-carrying His-residues in the most abundant species. Since myoglobin is a heme-binding protein it was additionally indicated that synthetic phosphorylation may retain protein folding targeting only structurally accessible His-residues. Insulin, βcasein and cytochrome C were phosphorylated on their His-residues and the corresponding peptides were detected in protein digest mixtures and in background of tryptically digested Escherichia coli lysate. In gel electrophoresis protocols, lengthy procedures at low pH such as staining reduced recovery. Synthetic phosphorylation of proteins and peptides with PPA allows the generation of suitable standard compounds for the systematic optimization of analytical protocols. All tested proteins responded to PPA treatment, partially even preserving tertiary structure. A distribution of modified protein forms was generated which could be subjected to further separation to isolate the fully phosphorylated species.