The majority of mitochondrial preproteins is targeted via N-terminal presequences that are cleaved upon import into the organelle. The essential mitochondrial processing protease (MPP) is assumed to cleave the majority of incoming precursors. However, only few substrate proteins of MPP have been experimentally determined and analysis of a broad range of substrates is missing so far. Here, we present the first systematic approach to identify substrate proteins of MPP. We used a temperature-sensitive mutant of the MPP subunit Mas1 grown at non-permissive temperature. Analysis of highly purified mitochondria by quantitative N-terminal ChaFRADIC led to the identification of 85 potential MPP substrate proteins. Deduction of the cleaved presequences shows that arginine in position -2 is the main determinant of MPP recognition. Interestingly, several non-processed proteins were increased in mas1 mutant mitochondria revealing a novel mitochondrial proteotoxic stress response that ultimately leads to an increased membrane potential presumably to balance/compensate the impairment of the major presequence processing peptidase.