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Abstract We introduce a new class of chemical probes for activity‐based sensing of proteases, termed cleavable, locked initiator probes (CLIPs). CLIPs contain a protease‐cleavable peptide linked between two programmable DNA strands—an “initiator” DNA and a shorter “blocking” DNA. These DNA sequences are designed to hybridize, creating a “locked” hairpin‐like structure. Upon proteolytic cleavage, the initiator strand is released, triggering the activation of CRISPR‐Cas12a enzymes and producing an amplified fluorescence response. CLIPs generate more than 20‐fold turn‐on signals at room temperature (25 °C), significantly outperforming commercial probes by yielding ∼40‐fold lower limits of detection (LOD) at 100‐fold lower concentrations. Their versatility enables the detection of various disease‐relevant proteases—including the SARS‐CoV‐2 main protease, caspase‐3, matrix metalloproteinase‐7, and cathepsin B—simply by altering the peptide sequence. Importantly, CLIPs detect cathepsin B in four different colorectal cancer cell lines, highlighting their clinical potential. Taken together, the sensitivity (LOD: ∼88 pM), selectivity, and rapid assay time (down to 35 min), combined with the ability to operate in complex biological media with minimal sample preparation, position CLIPs as powerful chemical tools for activity‐based sensing of functional enzymes.