The 13 amino acid EGFR-sequence AENAEYLRVAPQS-NH2 containing the in vivo autophosphorylated Tyr 1171, was synthesized by Fmoc continuous-flow SPPS with and without N-terminal Boc protection. In addition to the native sequence, peptides in which tyrosine was exchanged by serine and threonine were prepared. Global phosphorylation of the unprotected hydroxyl amino acids on the resin with di-tert-butyl-N,N-diethylphosphoramidite and 1H-tetrazole followed by in situ oxidation of the resulting phosphites with tert-butyl hydroperoxide or with dibenzoyl tetrasulfide resulted in the tyrosine-, serine- and threonine-phosphorylated and -thiophosphorylated sequences, respectively. The quality of the products after phosphorylation with N-terminal protection was better than without. Whereas the serine- and threonine-thiophosphate group was stable, tyrosine-thiophosphate turned out to be hydrolytically labile under acidic conditions. The rate of hydrolysis was determined with the tyrosine-thiophosphorylated model dipeptide Ac-Tyr-Gly-OH between pH 0.1 and 8. Hydrolysis was fastest at pH 3, with a half-time of 12.5 h at room temperature. The tyrosine-thiophosphate group was completely stable at pH 8.