Caspase-2 is an evolutionarily conserved but enigmatic protease whose biological role remains poorly understood. To date, research into the functions of caspase-2 has been hampered by an absence of reagents that can distinguish its activity from that of the downstream apoptotic caspase, caspase-3. Identification of protein substrates of caspase-2 that are efficiently cleaved within cells may also provide clues to the role of this protease. We used a yeast-based transcriptional reporter system to define the minimal substrate specificity of caspase-2. The resulting profile enabled the identification of candidate novel caspase-2 substrates. Caspase-2 cleaved one of these proteins, the cancer-associated transcription factor Runx1, although with relatively low efficiency. A fluorogenic peptide was derived from the sequence most efficiently cleaved in the context of the transcriptional reporter. This peptide, Ac-VDTTD-AFC, was efficiently cleaved by purified caspase-2 and auto-activating caspase-2 in mammalian cells, and exhibited better selectivity for caspase-2 relative to caspase-3 than reagents that are currently available. We suggest that this reagent, used in parallel with the traditional caspase-3 substrate Ac-DEVD-AFC, will enable researchers to monitor caspase-2 activity in cell lysates and may assist in the determination of stimuli that activate caspase-2 in vivo.