High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing high-throughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3β4 and hα4β2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC(50) values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHβE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.