For the first time, selective isolation of a target peptide based on the isoelectric point (pI) was achieved using a two-step electromembrane extraction (EME) approach with a thin flat membrane-based EME device. In this approach, step #1 was an extraction process, where both the target peptide angiotensin II antipeptide (AT2 AP, pI=5.13) and the matrix peptides (pI>5.13) angiotensin II (AT2), neurotensin (NT), angiotensin I (AT1) and leu-enkephalin (L-Enke) were all extracted as net positive species from the sample (pH 3.50), through a supported liquid membrane (SLM) of 1-nonanol diluted with 2-decanone (1:1 v/v) containing 15% (v/v) di-(2-ethylhexyl)-phosphate (DEHP), and into an aqueous acceptor solution (pH 1.80). In step #1, the cathode was located in the acceptor solution. Following step #1 (and prior to step #2), the pH of the acceptor solution was adjusted to pH 5.25, and the anode was located in the acceptor solution. Step #2 was a clean-up process to remove the matrix peptides with pI>5.13 (net positively charged) from the acceptor solution pH 5.25. During step #2, the target peptide was not net positively charged. This suppressed complex formation with negatively charged DEHP, and the target remained in the acceptor solution. The acceptor solution pH, the SLM composition, the extraction voltage, and the extraction time during the clean-up process (step #2) were important factors influencing the separation performance. An acceptor solution pH of 5.25 for the clean-up process slightly above the pI value (pH 5.13) was found to be optimal. Under the optimal conditions, 73% of AT2 AP (RSD 13%) and 48% of L-Enke (RSD 5%) were found in the solution after this two-step EME process, whereas the other three positively charged peptides were not detected. The observations above indicated that two-step EME may have a future potential for the fractionation of peptides and other ampholytic compounds based on their isoelectric points.