Relative potency (REP) estimates are widely used to characterize and compare the potency of a wide variety of samples analyzed using in vitro bioassays. Relative potency estimates are generally calculated as a simple ratio: the EC50 of a well-characterized standard divided by the EC50 of a sample. Such estimates are valid only when the dose-response curves for the sample and standard are parallel and exhibit the same maximum achievable response (efficacy). These conditions are often either violated or cannot be demonstrated. As a result, there is a need to calculate and present REPs in a manner that addresses the potential uncertainties caused by violation of the assumptions of parallelism and equal efficacy. Multiple point estimates, over the range of responses from EC20 to EC80, can be used to derive relative potency ranges (REP20–80 range). The width of a REP20–80 range is directly proportional to the degree of deviation from parallelism between sample and standard dose-response curves. Thus, REP20–80 ranges both test the assumption of parallelism and characterize the amount of uncertainty in an REP estimate resulting from deviation from parallelism. Although uncertainties due to unequal efficacy cannot be easily characterized mathematically, a systematic method for evaluating sample efficacy has been developed into a framework to guide the derivation and application of REP estimates based on in vitro bioassay results. Use of the systematic framework and REP20–80 ranges was illustrated using three sample data sets. It is hoped that the framework and discussion presented will facilitate the use of bioassay-derived REP estimates to characterize samples of both known and unknown composition without ignoring the assumptions underlying REP estimation.