Abstract Introduction Questionnaires that inquire about REM sleep behavior disorder are typically validated by reference to polysomnographic findings of REM sleep without atonia. We sought validation for such a questionnaire by examining observed REM-based dream enactment over two nights in a sleep laboratory. Methods Participants with alpha-synucleinopathic disease underwent two consecutive nights of polysomnography attended by a single sleep technologist who was instructed to observe for and document any abnormal behaviors occurring out of sleep. We evaluated University of Michigan REM Behavior Disorder Questionnaire responses completed by either the participant or bed-partner prior to polysomnographic recording and analyzed associations between questionnaire responses and observational data in the sleep laboratory. We subsequently cross-validated the technologist’s observations with hypnogram tracings from nights when dream enactment was observed to determine whether the timing of observed behavior aligned with the timing of REM sleep. Results We studied 92 participants [idiopathic Parkinson’s disease, n = 67; Dementia with Lewy bodies, n = 14; Multiple Systems Atrophy, n = 3; Idiopathic REM sleep behavior disorder, n = 8]. Of 92 participants studied, 30 (32.61%) exhibited dream enactment during REM sleep observed on either night of polysomnographic recording (11 on both nights, 19 on one night). There was a statistically significant difference (p = 0.041 by 2-tailed test) in mean questionnaire scores between participants with observed dream enactment during REM sleep (mean score 0.43; [95% CI mean 0.34-0.52]) and those without dream enactment on either night (mean score 0.32; [95% CI mean 0.26-0.38]). When limiting the sample to participants whose questionnaire responses were derived from the bed-partner (n = 74), the difference approached, but did not reach, statistical significance (p = 0.066). Conclusion These data provide further validation of the University of Michigan REM Behavior Disorder Questionnaire as a useful tool in evaluating REM-based dream enactment behavior in patients with alpha-synucleinopathic disease. Support (If Any)