We applied PCT-DIA on a total of 1161 nodules from 1133 patients using either tissue cores (1 mm diameter; 0.5–1 mm depth) punched from regions of interest marked on retrospective FFPE tissue blocks or prospective cytology specimens from FNA aspirates. The samples comprise (i) a discovery set of FFPE samples from Singapore General Hospital (n = 579 nodules) where histopathological diagnoses were confirmed on central review by a board-certified pathologist; and independent test sets from twelve hospitals in China and Singapore consisting of (ii) retrospective test sets of FFPE samples (n = 288 nodules) with the same histopathological assessment and classification as the discovery sample set, and (iii) a prospective test set of FNA biopsies (n = 294 nodules) which were additionally scored by the Bethesda System for reporting thyroid cytopathology.