Regulation of protein homeostasis is crucial for the appropriate functioning and maintenance of every living cell. Therefore, spatial and functional control over protein abundance and activity is tightly regulated. One key regulatory system of protein homeostasis is the Ubiquitin Proteasome System (UPS) which is heavily involved in targeted protein degradation of e.g. unneeded or misfolded proteins. The N-end rule pathway, as a specialized branch of the UPS, correlates the in vivo half-life of a protein directly to its presented N-terminus. Until now, only very few components or substrates of the N-end rule pathway have been described in plants. Through application of comparative 2D gel electrophoresis and shotgun analysis, more than 50 proteins identified, which showed a higher abundance in mutants of the N-end rule pathway in comparison to their wild type counterpart. After validation of the in vivo N-termini, two potential new substrates were further characterized by interaction experiments through various means including Arabidopsis protoplasts. This study suggest that the cysteine protease RD21a is the first potential substrate of the arginylation branch of the N-end rule pathway which doesn’t start with MC at its N-terminus. Furthermore, it was demonstrated, that the protein RIN4, which has long been discussed as an N-end rule substrate with common databases such as TAIR even listing it as such, is probably not a substrate.