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The assessment of pain in patients with brain injury is challenging due to impaired ability to communicate. We aimed to test the reliability and validity of the critical-care pain observation tool (CPOT) and the bispectral index (BIS) for pain detection in critically brain-injured patients.This prospective observational study was conducted in a neurosurgical intensive care unit in a University-Affiliated Hospital. Adult brain-injured patients undergoing mechanical ventilation were enrolled. Nociceptive (endotracheal suctioning) and non-nociceptive (gentle touching) procedures were performed in a random crossover fashion. Before and immediately after the procedure, CPOT was evaluated by 2 residents and 2 chief nurses, and BIS was documented. The ability to self-report pain was also assessed. The inter-observer reliability of CPOT was analyzed. The criterion and discriminant validities of the CPOT and the BIS were tested.During the study, we enrolled 400 brain-injured patients. The ability to self-report pain was maintained in 214 (54%) and 218 (55%) patients during suctioning and gentle touching, respectively. The intraclass correlation coefficients (95% confidence interval) for inter-observer reliability of CPOT ranged from 0.86 (0.83–0.89) to 0.93 (0.91–0.94). Using self-reported pain as the reference, the area under the receiver operating characteristic curve (95% confidence interval) was 0.84 (0.80–0.88) for CPOT and 0.76 (0.72–0.81) for BIS. When the 2 instruments were combined as either CPOT ≥2 or BIS ≥88 after the procedure, the sensitivity and specificity were 0.90 (0.85–0.93) and 0.59 (0.52–0.66), respectively; and when the 2 instruments were combined as both CPOT ≥2 and BIS ≥88, the sensitivity and specificity were 0.62 (0.55—0.68) and 0.89 (0.83–0.93). Both CPOT and BIS increased significantly after suctioning (all P < .001) but remained unchanged after gentle touching (P ranging from .06 to .14).Our criterion and discriminant validity results supported the use of CPOT and BIS to detect pain in critically brain-injured patients. Combining use of CPOT and BIS in different ways might provide comprehensive pain assessment for different purposes.