Membrane-bound transporter proteins are involved in cell signal transduction and metabolism as well as influencing key pharmacological properties such as drug bioavailability. The functional activity of transporters that belong to the group of electrically active membrane proteins can be directly monitored using the solid-supported membrane-based SURFE(2)R™ technology (SURFace Electrogenic Event Reader; Scientific Devices Heidelberg GmbH, Heidelberg, Germany). The method makes use of membrane fragments or vesicles containing transport proteins adsorbed onto solid-supported membrane-covered electrodes and allows the direct measurement of their activity. This technology has been used to develop a robust screening compatible assay for Complex I/Complex III, key components of the respiratory chain in 96-well microtiter plates. The assay was screened against 1,000 compounds from the ComGenex Lead-like small molecule library to ascertain whether mitochondrial liabilities might be an underlying, although undesirable feature of typical commercial screening libraries. Some 105 hits (compounds exhibiting >50% inhibition of Complex I/Complex III activity at 10 μM) were identified and their activities were subsequently confirmed in duplicate, yielding a confirmation rate of 68%. Analysis of the confirmed hits also provided evidence of structure-activity relationships and two compounds from one structural class were further evaluated in dose-response experiments. This study provides evidence that profiling of compounds for potential mitochondrial liabilities, even at an early stage of drug discovery, may be a necessary additional quality filter that should be considered during the compound screening and profiling cascade.