Phosphorylation of amino acid residues in proteins and peptides is a very important modification playing a vital role in living organisms. That is a reason why it is so important to have an efficient tool for isolation and identification of phoshorylated peptides and proteins. The efficient technique combining IMAC (immobilized metal ion affinity chromatography) and RP-HPLC (HPLC on reversed phase) for determination of phosphorylated peptides in proteolytic digest was verified on model proteins with well defined phosphorylations. Peptide fragments created by digestion of β-casein from bovine milk with trypsin and swine pepsinogen A digested with α-chymotrypsin were separated in the first step by IMAC. Peptides containing phosphate group(s) were bound to ferric ions (Fe3+) immobilized on iminodiacetate (IDA)–Sepharose 6B. Phosphorylated peptides were then released by 0.1 M sodium phosphate (pH 5.0). Reversed phase high performance chromatography (RP-HPLC) was applied as the subsequent step for the separation of individual phosphopeptides from the mixture obtained by immobilized metal ion affinity chromatography. The developed method was then applied on pepsinogen A digests from stomach mucosa of patients suffering with gastric cancer, and pepsinogen A digests of patients suffering with peptic ulcer (without cancer). Significantly, higher amounts of phosphopeptides were found in samples obtained from patients with gastric cancer. Thus, it seems evident that the higher level of pepsinogen A phosphorylation seems to be associated with gastric cancer. The method developed represents a reliable tool for the study of phosphorylation degree of proteins in general. In this particular case, it could serve, potentially, for an early diagnosis of gastric cancer.