2-DE is still a relatively cumbersome and labor intensive method. Given the successful cysteinyl protection concept with hydroxyethyl disulfide (specific oxidation) during the first dimension separation, the possibility for a simplified equilibration procedure was investigated. This was achieved by maintaining the S-mercaptoethanol modified cysteinyls throughout the 2-D workflow including second dimension separation, spot handling, protein digestion, and protein identification. The traditional equilibration protocol encompassing thiol reduction and alkylation was compared with a one-step protocol employing continuous exposure to hydroxyethyl disulfide. Both equilibration protocols gave equally well-resolved spot maps with analytical protein loads regardless of IPG strip pH range. Using preparative protein loads, narrow range IPG strips gave comparable results for the two protocols while preparative load on wide range IPG strips was the only condition where classical reduction/alkylation outperformed hydroxyethyl disulfide equilibration. Moreover, with analytical protein loads, the hydroxyethyl disulfide equilibration time could be significantly reduced without apparent loss of spot map quality or quantitative protein transfer from the first- to the second dimension gel. MALDI-TOF mass spectrometric protein identification was successfully performed with either iodoacetamide or hydroxyethyl disulfide as the cysteine modifier, yielding comparable identification results with high confidence in protein assignment, sequence coverage, and detection of cysteine-containing peptides. The results provide a novel and simplified protocol for 2-DE where the concept of hydroxyethyl disulfide as the cysteinyl protecting agent is extended to cover the entire 2-D work flow.