Caspases are a family of cysteine-aspartyl proteases that are well recognized for their essential roles in apoptosis and inflammation. Recently, caspases have also been linked to the promotion of other biologically important phenomena, such as cellular differentiation and proliferation. Dysregulation of the multifaceted and indispensable activities of caspases has been globally linked to several diseases, including cancer and neurodegenerative disorders; however, the specific caspase members responsible for these diseases have yet to be assigned. Activity-based probes (ABPs) and peptide-based inhibitors are instrumental in the detection and control of protease activity and serve as alternative methods to genetic approaches. Such molecules aid in the interrogation of specific proteases within cellular and animal models as well as help elucidate aberrant proteolytic function correlated to disease phenotypes. No ABPs or inhibitors have been discovered that specifically target one of the eleven human caspases in a cellular context. Therefore, ascribing distinct contributions to an individual caspase activity within naturally occurring biological systems is not possible. Herein, we describe a peptide series optimized for the selective detection and inhibition of active caspase-3 in cells. These compounds exhibit low nanomolar potency against caspase-3 with >120-fold selectivity over caspase-7 which shares 77% active site identity. Our ability to individually target wild-type active caspase-3 for detection and cell permeable inhibition is a valuable proof-of-concept methodology that can be readily employed to probe the significance of caspase-3 in apoptosis, neurological disorders, cardiovascular diseases, and sepsis.