Interestingly, we recently showed that only AF4NMLL but not the reciprocal Cycloheximide cost translocation product, MLLNAF4, lacking the Taspase1 cleavage site, can cause proB ALL in a murine model. Thus, proteolytic cleavage of MLL-fusion proteins by Taspase1 is considered a critical step for MLL-mediated tumorigenesis, although the molecular details are not yet resolved. Besides Taspase1’s role in leukemogenesis the protease was suggested to be also overexpressed solid tumors. In this respect, recent data indicate that also other regulatory proteins, such as the precursor of the Transcription Factor IIAor Drosophila HCF, are Taspase1 targets. Hence, there is an increasing interest in defining novel Taspase1 targets. However, the molecular mechanisms how Taspase1 affects biological functions through site-specific proteolysis of its substrates and what other cellular programs are regulated by Taspase1’s degradome under normal or pathophysiological conditions is completely unknown. Besides genetic instruments, chemical decoys allowing the targeted inhibition/activation of proteins are powerful tools to dissect complex biological pathways. Small molecules that allow a chemical knock out of a cellular reaction or a cell phenotype can be selected by phenotypic screens, and used as molecular tools to identify previously uncharacterized proteins and/or molecular mechanisms. Hence, chemogenomics as studying the interaction of biological systems with exogenous small molecules, i.e., analyzing the intersection of biological and chemical spaces, seems an attractive approach to also dissect Taspase1 functions. Unfortunately, Taspase1��s catalytic activity is not affected by common protease CHIR-99021 GSK-3 inhibitor inhibitors and no small molecule inhibitors for this enzyme are currently available to dissect Taspase1’s function in vivo. As biochemical data or potential drugs must be effective at the cellular level, reliable cell-based assaysfor Taspase1 are urgently needed. Often, redistribution approaches, as cell-based assay technology that uses protein translocation as the primary readout have been used to study the activity of cellular signaling pathways. Protein targets are labeled with autofluorescent proteins and are read using high-throughput, microscope-based instruments. Although, protein translocation assays have the potential for high-content, high-throughput screeningapplications, such assays are generally not used for proteases. Here, the spatial and functional division into the nucleus and the cytoplasm was exploited to design a translocation-based Taspase1-biosensor assay. The CBA was adapted on a HTS platform, employed to identify potential Taspase1 small molecule inhibitors, and was used to study Taspase1 function in living cells. The robust performance of the TS-Cl2+CBA met critical requirements for high content screening: the biosensor was nontoxic, localized to the cytoplasm in the absence of ectopically expressed Taspase1, and efficiently accumulated in the nucleus following Taspase1-specific cleavage. Hence, we tested whether the assay can also be used on a high-throughput microscopy based screening platform. As cell lines inducibly expressing biosensors may facilitate certain HCS/HTS applications, we generated stable Tet-off TSCl2 + TRE cell lines. The tetracycline -regulated system has been used successfully in various applications. Whereas expression of TS-Cl2 + TRE was blocked in the presence of doxycycline, Dox removal induced TS-Cl2+ TRE expression. Cleavage of TS-Cl2+TRE by the endogenous Taspase1 subsequently resulted in nuclear accumulation of the biosensor.