Studying biological processes on the level of live cells with the help of biocompatible reac-tions has tremendously advanced our understanding of basic biology. However, the great complexity of many human pathologies such as cancer, diabetes and neurodegenerative diseases requires new tools that would allow investigation of biological processes throughout the organism. The 2-cyanobenzothiazole (CBT)-based ligation reaction has received a recent interest in the chemical biology community. It has been reported in the literature for various applications, ranging from fluorescent labelling of proteins to nanostructures formation, and, most importantly, the reaction was shown to proceed in cells. This selective reaction between D-cysteine and hydroxy-CBT (HO-CBT) or amino-CBT (H2N-CBT), also named as split luciferin reaction, generates as product a D-luciferin analogue, one of the most commonly used substrates for bioluminescence imaging (BLI). Therefore, the split luciferin reaction has high potential for BLI applications. In this work, we have shown that production of a luciferin substrate via the split luciferin reaction can be visualized in live mice using BLI. Furthermore, the split luciferin approach allows interrogation of target tissues using a masking approach, where D-luciferin is formed only under certain conditions. This reaction was successfully applied to real-time non-invasive imaging of apoptosis, associated with caspase 3/7 activity. Caspase-dependent release of free D-cysteine from a caspase 3/7 specific peptide substrate allowed selective reaction with H2N-CBT in vivo to form 6-amino-D-luciferin with subsequent light emission in the presence of the firefly luciferase enzyme. Importantly, this strategy was found to be superior to the use of the commercially available DEVD-aminoluciferin substrate for imaging caspase 3/7 activity. The same methodology was extended to imaging activity of other caspases as well as thrombin enzyme in an in vitro set-up. Furthermore, the split luciferin approach enables dual imaging, where each reaction partner would be individually caged to report on separate biological events. This approach was used for simultaneous imaging of caspase 3 and β-galactosidase in vitro, validating the use of the split luciferin reaction for imaging multiple processes. Moreover, the split luciferin reaction was also successfully applied to both quantification of Neutrophil Elastase activity in vitro and real-time non-invasive imaging of Neutrophil Elastase in an in vivo inflammation model. Altogether, the present study suggests that the split luciferin approach is an efficient and versatile tool for in vivo applications.