Few individuals with substance use disorders limit their intake to one substance of abuse; however, many studies focus on a single substance. Unfortunately, the optimal method to determine the principal substance is unclear. In particular, this issue is problematic in patients with co-occurring psychiatric illness, who commonly use multiple substances. Hence we compared three methods for assessing the principal substance of abuse in 150 subjects with bipolar disorder and substance dependence: 1) the Addiction Severity Index interview, 2) a self-administered questionnaire, and 3) the most frequently used substance. While most subjects were concordant on the interview and the other two methods, we found substantial disagreement (9.3% between the interview and the questionnaire, and 12.7% between the interview and the most frequently used substance) and partial agreement (14.0%). These findings from a comorbid population demonstrate that different methods to assess principal substance of abuse could lead to different conclusions about treatment outcomes. Hence studies of comorbid patients may benefit from 1) using more than one method to assess principal substance and 2) reporting use of all substances as well as a targeted substance.