OBJECTIVE: To examine the validity of the Disruptive Behavior Diagnostic Observation Schedule (DB-DOS), a new observational method for assessing preschool disruptive behavior. METHOD: A total of 327 behaviorally heterogeneous preschoolers from low-income environments comprised the validation sample. Parent and teacher reports were used to identify children with clinically significant disruptive behavior. The DB-DOS assessed observed disruptive behavior in two domains, problems in Behavioral Regulation and Anger Modulation, across three interactional contexts: Examiner Engaged, Examiner Busy, and Parent. Convergent and divergent validity of the DB-DOS were tested in relation to parent and teacher reports and independently observed behavior. Clinical validity was tested in terms of criterion and incremental validity of the DB-DOS for discriminating disruptive behavior status and impairment, concurrently and longitudinally. RESULTS: DB-DOS scores were significantly associated with reported and independently observed behavior in a theoretically meaningful fashion. Scores from both DB-DOS domains and each of the three DB-DOS contexts contributed uniquely to discrimination of disruptive behavior status, concurrently and predictively. Observed behavior on the DB-DOS also contributed incrementally to prediction of impairment over time, beyond variance explained by meeting DSM-IV disruptive behavior disorder symptom criteria based on parent/teacher report. CONCLUSIONS: The multidomain, multicontext approach of the DB-DOS is a valid method for direct assessment of preschool disruptive behavior. This approach shows promise for enhancing accurate identification of clinically significant disruptive behavior in young children and for characterizing subtypes in a manner that can directly inform etiological and intervention research.