The tendency to interpret ambiguous everyday situations in a relatively negative manner (negative interpretation bias) is central to cognitive models of depression. Limited tools are available to measure this bias, either experimentally or in the clinic. This study aimed to develop a pragmatic interpretation bias measure using an ambiguous scenarios test relevant to depressed mood (the AST-D). In Study 1, after a pilot phase (N = 53), the AST-D was presented via a web-based survey (N = 208). Participants imagined and rated each AST-D ambiguous scenario. As predicted, higher dysphoric mood was associated with lower pleasantness ratings (more negative bias), independent of mental imagery measures. In Study 2, self-report ratings were compared with objective ratings of participants' imagined outcomes of the ambiguous scenarios (N = 41). Data were collected in the experimental context of a functional Magnetic Resonance Imaging scanner. Consistent with subjective bias scores, independent judges rated more sentences as negatively valenced for the high versus low dysphoric group. Overall, results suggest the potential utility of the AST-D in assessing interpretation bias associated with depressed mood.