Human intestinal permeability is a key determinant of the oral fraction absorbed (fa) of active pharmaceutical ingredients (APIs). This study evaluated the ability of an in-house canine Mdr1 (cMdr1) knockout (KO) Madin-Darby Canine Kidney (MDCK) cell line to correlate in vitro apparent permeability (Papp) with human small intestinal permeability (Peff). In vitroPapp values of 16 reference compounds with high, medium, or low permeabilities were measured in the in-house cMdr1 KO MDCK protocol under pH gradient (6.5 ⇒ 7.4) and pH equivalent conditions (7.4 ⇒ 7.4) and correlations with human Peff were established (R2 > 0.8). The correlations were subsequently used to estimate Peff and fa for six test APIs: acetaminophen, voriconazole, fedratinib, voxelotor, lemborexant, and istradefylline. The results for these APIs were compared against literature and permeability data from other methods routinely used in drug discovery and development. The projected Peff and fa values for the test APIs aligned well with literature permeabilities derived using other methods and clinical pharmacokinetic studies, respectively. This work highlights the usefulness of cMdr1 KO MDCK cells in permeability classification, especially for highly permeable APIs, and supports its broader use in both research and regulatory contexts.