Taspase1 is a threonine protease responsible for cleaving intracellular substrates. As such, (de)regulated Taspase1 function is expected not only to be vital for ordered development but may also be relevant for disease. However, the full repertoires of Taspase1 targets as well as the exact biochemical requirements for its efficient and substrate-specific cleavage are not yet resolved. Also, no cellular assays for this protease are currently available, hampering the exploitation of the (patho)-biological relevance of Taspase1. Here, we developed highly efficient cell-based translocation biosensor assays to probe Taspase1 trans-cleavage in vivo. These modular sensors harbor variations of Taspase1 cleavage sites and localize to the cytoplasm. Expression of Taspase1 but not of inactive Taspase1 mutants or of unrelated proteases triggers proteolytic cleavage and nuclear accumulation of the biosensors. Employing our assay combined with scanning mutagenesis, we identified the sequence and spatial requirements for efficient Taspase1 processing in liquid and solid tumor cell lines. Collectively, our results defined an improved Taspase1 consensus recognition sequence,, allowing the first genome-wide bioinformatic identification of the human Taspase1 degradome. Among the 27 most likely Taspase1 targets are cytoplasmic but also nuclear proteins, such as the upstream stimulatory factor 2 (USF2) or the nuclear RNA export factors 2/5 (NXF2/5). Cleavage site recognition and proteolytic processing of selected targets were verified in the context of the biosensor and for the full-length proteins. We provide novel mechanistic insights into the function and bona fide targets of Taspase1 allowing for a focused investigation of the (patho)biological relevance of this type 2 asparaginase.By cleaving proteins, proteases are involved in the control of a large number of key physiological processes such as development, metabolism, tissue remodeling, cell proliferation, and apoptosis (1-3). Protease signaling therefore differs from the majority of other signaling pathways by being mostly irreversible (3). Protease signaling is strictly regulated, and the deregulation of protease activity can contribute to various pathologies, including cancer (3).The human Taspase1 gene encodes a protein of 420 amino acids (aa), 4 which is the proenzyme of Taspase1. It belongs to a family of enzymes possessing an asparaginase-2 homology domain. In the MEROPS database, Taspase1 is found as T02.004, classifying this protein as a class PB, subclass PB(T), and T2 family protease. In contrast to the other cis-active type 2 asparaginases, such as amidohydrolases, L-asparaginase, and glycosylasparaginase, only Taspase1 is able to cleave other substrates in trans (4). Therefore, Taspase1 represents a distinct class of proteolytic enzymes. Taspase1-mediated cleavage of proteins follows distinct aspartate residues, suggesting that Taspase1 evolved from hydrolyzing asparagines and glycosylasparagines to recognize a conserved peptide motif with an aspartate at the P1 ...