The absorptive (AQ) and secretory (SQ) quotients have been proposed as a novel experimental approach to quantify the modulation of intestinal absorption and secretion by P-glycoprotein (Pgp). Because these unidirectional assays inherently assess for the impact of Pgp, conclusions as to whether a compound is a Pgp substrate will be made from the data. Therefore, the objective of this study was to establish the relationship between AQ/SQ and the bidirectional efflux assay and to derive criteria to classify a compound as a Pgp substrate. AQ and SQ parameters were calculated for 331 compounds that had previously been evaluated in the bidirectional assay and the concordance of Pgp substrate classification between these methods assessed by establishing AQ/SQ criteria of increasing magnitude. The AQ and SQ values correctly identified 80 and 85% of the compounds as Pgp substrates/nonsubstrates relative to the bidirectional efflux assay. This study demonstrates that the optimal AQ and SQ value to classify compounds as Pgp substrates was 0.3 and provides a basis to deploy unidirectional efflux assays in the early stages of drug discovery, which would benefit from the twofold increase in throughput over current bidirectional transport assays.