From 1994 to 2000, 154 sites belonging to the grayling and trout zones were used to develop a fish-based multimetric Index of Biotic Integrity (IBI) as a tool to assess upstream brooks in Flanders (Belgium). All sites had a slope of at least 3‰ and a maximum width of 4.5 m. Fish assemblages were surveyed by electrofishing. From 28 candidate metrics 9 metrics were selected using ecological criteria and statistical analyses. The IBI was defined as the average of these 9 metrics after scoring them as 1, 3 or 5 by a modified trisection method and five integrity classes were defined to comply with the European Water Framework Directive (WFD). The IBI was tested internally by comparison with habitat quality scores at sites. A similar comparison was done using an independent set of data from sites of known ecological quality. Our IBI clearly distinguished good sites from disturbed sites and heavily disturbed sites from moderately disturbed sites, thereby meeting the criteria imposed by the WFD.