Background: Assessing pain of critically ill patients with brain injuries who are unable to communicate is a challenge. Current behavioral scales are limited in accurate pain assessments for this population. Aims: This study sought to investigate the behavioral and physiological responses induced by routine painful procedures in patients with brain injuries who are unable to communicate. Methods: Using a repeated-measure within-subject observational study design, 12 participants admitted to an intensive care unit were observed before, during, and 15 minutes after a nonnociceptive (noninvasive blood pressure measurement) procedure and three nociceptive (suctioning, turning, and trapezius pinch) procedures. During each assessment, patients’ behavioral and physiological responses were observed using video cameras and bedside monitors. Results: In the overall behavioral responses to the nociceptive procedures, clenched teeth with tense jaw, frowning, orbit tightening, closing of eyes, eye movement, fixation-staring, flushing, flexion withdrawal of arm, flexion withdrawal of leg, muscle rigidity, twitching, and coughing were more frequently observed during procedures than before and after procedures (p < .01). Regarding physiological responses, significant increases in systolic blood pressure, diastolic blood pressure, mean arterial pressure, heart rate, and respiratory rate were identified across assessments (p < .001). Conclusions: The findings can be used as a basis for pain assessment and the development of pain assessment tools for brain-injured patients who are unable to communicate. However, since physiological responses may be influenced by various factors besides pain, physiological changes may be used as a sign of the need for pain assessment rather than being used alone as a basis for pain assessment.