Cathepsin X/Z/P is protease in the lysosomal cysteine cathepsin family that exhibits unique monocarboxypeptidase activity. Its expression has been associated with several cancer types and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Like most proteases, cathepsin X activity is subject to complex post-translational regulation. It is synthesised as an inactive zymogen that must be cleaved by other proteases to permit activation, and its activity can be further controlled by endogenous inhibitors. Thus, measures of protein abundance do not reflect the pool of proteolytically active cathepsin X. Advances in our understanding of the function of cathepsin X have been hindered by a lack of available tools that can specifically measure its activity. We therefore developed a series of new fluorescent activity-based probes that can bind to cathepsin X in an activity-dependent manner. These probes exhibit improved specificity and potency for cathepsin X compared to previously reported methods. We demonstrated the ability of these probes to detect the activity of cathepsin X in cell and tissue lysates, in live cells and in vivo, and to localise active cathepsin X in mouse tissues by confocal microscopy. We have applied our most sensitive and specific probe to study cathepsin X activation in oral squamous cell carcinoma. Biopsies from oral squamous cell carcinomas exhibited a significant increase in cathepsin X activity compared to patient-matched normal tongue tissue. Application of these probes, in combination with specific cathepsin X inhibitors, knockout mice, and oral cancer models, will advance our understanding of cathepsin X in oral cancer pathogenesis.