Abstract A dramatic increase in the number of patients being diagnosed with incidental thyroid nodules through different diagnostic imaging modalities has created a unique opportunity to detect and remove lesions at early stages of malignancy. This also creates a critical need for improved diagnostics, as 80% of patients currently undergo unnecessary thyroidectomies because their nodules were benign. Current standard of care includes collecting a tissue biopsy by ultrasound- guided fine needle aspiration: these samples are tested by cytology, which looks for aberrant cells, and for genetic risk markers. These diagnostic tests perform poorly, consequently a third of all nodules are still classified as indeterminate and are removed for a pathological diagnosis. We hypothesized that dysregulated proteolysis, a type of enzyme activity that is a hallmark of invasive cancer, might yield discriminating levels of activity in FNA tissue from benign and malignant nodules. The Alaunus Biosciences, Inc. diagnostic pipeline takes advantage of a substrate profiling technology developed in the Craik Laboratory at UCSF, a platform that allows innovative proteomics-based biomarker discovery. Using this technology, we have previously identified and characterized a proprietary set of protease activities that are significantly increased in pre-malignant pancreatic cysts; we then developed fluorogenic substrate assays to monitor these activities using small volumes of liquid biopsy (cyst fluid). In this Phase I proposal we aim to apply our discovery pipeline to a retrospective cohort of pathologically-confirmed thyroid cancer and matched normal tissue, to identify protease activities that are specific to malignant tissue. In Aim 1, we will profile thyroid tumor-specific proteolytic signatures, and identify candidate enzyme markers responsible for the observed proteolytic activity. In Aim 2, we will design and rationally optimize specific fluorogenic substrates for these proteases that can be used to rapidly detect malignant nodules in FNA samples. Lead candidate substrates will proceed to a robust validation of their clinical utility as diagnostic tools, by benchmarking a prototype assay against the current clinical standard guidelines. Our goal is to develop a rapid assay that improves patient stratification over current standard diagnostic markers and guides clinical decision-making to avoid unnecessary surgical intervention. If successful, this proposal will lay the groundwork for an actionable diagnostic test that will enable improved risk stratification of indeterminate thyroid nodules, and transform the clinical management of these challenging lesions.