Two predictive tools have been proposed by Austin et al. (Drug Metab Dispos 30:1497-1503, 2002) and Hallifax and Houston (Drug Metab Dispos 34:724-726, 2006) to estimate the fraction unbound in the incubation (fuinc). The current study was undertaken to elucidate the relative utility of these prediction tools over a range of drug lipophilicity and microsomal protein concentration. The fuinc data set (n = 127) comprised 35 drugs determined experimentally in this study and 92 collated from Austin and Hallifax data. The observed fuinc values at three microsomal concentrations were compared with the estimates obtained using the Austin and Hallifax equations. In addition, the impact of variability in the logP on the fu inc predictions was assessed. The current analysis highlights the importance of accurate estimation of lipophilicity for the prediction of the fuinc, regardless of the prediction equation used. Both equations represent useful tools for estimation of fuinc for low lipophilicity drugs (logP/D = 0-3), especially at low microsomal protein concentration. However, the accuracy of fuinc predictions of highly lipophilic drugs was poor for both equations, implying that fuinc should be experimentally confirmed for drugs with logP/ D ≥ 3, unless the microsomal protein concentration is as low as 0.1 mg/ml, in which case a cutoff of logP/D ≥ 5 can be applied. A significant difference in the predictions by the two proposed tools was observed in the area of intermediate lipophilicity (logP/D = 2.5-5), where the Hallifax equation provided more accurate fuinc predictions on average, irrespective of the microsomal protein concentration investigated. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics.