Recently the clinical importance of human organic cation transporters 1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition, for example, clearance, toxicity, and drug-drug interactions, have been highlighted [Annu. Rev. Pharmacol. Toxicol. 2012, 52, 249-273; Nat. Rev. Drug Discovery 2010, 9 (3), 215-236]. Consequently, there is an extensive need for experimental assessment of structure-transport relationships as well as tools to predict drug uptake by these transporters in ADMET (absorption, distribution, metabolism, excretion, toxicity) investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using transfected HEK293 cells. For the first time, an extensive data set comprising uptake of 354 compounds is presented. As expected, there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular, a chemical series of phenylthiophenecarboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed: molecular volume was the most important determinant of hOCT1 substrates, whereas H-bonding parameters like polar surface area (PSA) dominated for hOCT2.