A reversed phase high pressure liquid chromatography method was developed for determination of in vitro refolding and cleavage kinetics for the N pro autoprotease fusion peptide EDDIE-pep6His using a TSK Super-Octyl column with a segmented acetonitrile gradient. Self-cleaving fusion proteins such as N pro autoprotease fusion proteins consist of the single autoprotease N pro and a target peptide or a target protein as fusion partner. Hence, three protein species are present after self-cleavage: the target peptide or protein, the single N pro autoprotease and, in case of incomplete cleavage, residual N pro fusion protein. Thus, for an accurate analysis the method must be standardized for three components in the presence of host cell impurities. For method validation, protein standards of EDDIE-pep6His and the single N pro autoprotease EDDIE were prepared from inclusion bodies (IBs) by ion exchange, immobilized metal ion affinity, size exclusion, and reversed phase chromatography. A linear correlation was obtained for EDDIE-pep6His and EDDIE in the range from 95 to 730μg/ml with a lower limit of quantification (LLOQ) and a lower limit of detection (LLOD) of 34.5 and 11.4μg/ml, respectively, for EDDIE-pep6His and 39.6 and 13.1μg/ml, respectively, for EDDIE. Finally, a fully automated batch refolding of EDDIE-pep6His from IBs was performed to demonstrate the applicability of this method. It was shown that the initial EDDIE-pep6His concentration in the refolding solution decreased from 194.3 to 83.8μg/ml over a refolding time of 385min resulting in a final refolding and cleavage yield of 50%.