The level of sampling effort required to characterize fish assemblage condition in a river for the purposes of bioassessment may be estimated via different approaches. However, the goal with any approach is to determine the minimum level of effort necessary to reach some specific level of confidence in the assessment. In the Ohio River, condition is estimated and reported primarily at the level of pools defined by lock and dam structures. The goal of this study was to determine the minimum level of sampling effort required to adequately characterize pools in the Ohio River for the purpose of bioassessment. We followed two approaches to estimating required sampling effort using fish assemblage data from a long-term intensive survey across a number of Ohio River pools. First, we estimated the number of samples beyond which variation in the multimetric Ohio River Fish Index (ORFIn) leveled off. Then, we determined the number of samples necessary to collect approximately 90% of the fish species observed across all samples collected within the pool. For both approaches, approximately 15 samples were adequate to reduce variation in IBI scores to acceptable levels and to capture 90% of observed species in a pool. The results of this evaluation provide a basis not only for the Ohio River Valley Water Sanitation Commission (ORSANCO) but also states and other basin commissions to develop sampling designs for bioassessment that ensure adequate sampling of all assessment units.