Despite the key role of proteolysis in biological processes, such as plant immunity, seed development, and abiotic stress responses, our knowledge on the identity of natural protease substrates in plants remains scarce. In the genome of the model plant Arabidopsis thaliana, for instance, approximately 700 genes code for proteases. However only a few natural substrates have been identified. Metacaspases are distant relatives of metazoan caspases, found in plants, fungi and protists. We report on the proteom-wide substrate identification of a plant protease using positional proteomics and mass spectrometry. We used the N-terminal COFRADIC technology to explore the substrate repertoire of Arabidopsis thaliana metacaspase-9 during early seedling development. This analysis provided additional insights into AtMC9 substrate cleavage specificity and revealed metacaspase functions other than those related to programmed cell death.