Regulatory agencies have recently issued drug-drug interaction guidelines, which require determination of plasma protein binding (PPB). To err on the conservative side, the agencies recommend that a 0.01 lower limit of fraction unbound (fu) be used for highly bound compounds (>99%), irrespective of the actual measured values. While this may avoid false negatives, the recommendation would likely result in a high rate of false positive predictions, resulting in unnecessary clinical studies and more stringent inclusion/exclusion criteria, which may add cost and time in delivery of new medicines to patients. In this perspective, we provide a review of current approaches to measure PPB, and important determinants in enabling the accuracy and precision in these measurements. The ability to measure fu is further illustrated by a cross-company data comparison of PPB for warfarin and itraconazole, demonstrating good concordance of the measured fu values. The data indicate that fu values of ≤0.01 may be determined accurately across laboratories when appropriate methods are used. These data, along with numerous other examples presented in the literature, support the use of experimentally measured fu values for drug-drug interaction predictions, rather than using the arbitrary cutoff value of 0.01 as recommended in current regulatory guidelines.