Introduction: A Tecan-based enzyme inhibition assay has been developed for the determination of atorvastatin-derived ‘active’ and ‘total’ (active inhibitors plus atorvastatin lactone and other potential inhibitors following base hydrolysis) 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor concentrations in human plasma. Atorvastatin is an inhibitor of HMG-CoA reductase, which is a key rate-limiting enzyme in the cholesterol biosynthesis. Previously, atorvastatin-derived HMG-CoA reductase inhibitors were measured via enzyme inhibition assays by manual operation. Methods: In this work, an enzyme assay procedure based on 8-tip Tecan robotics and set-up in a 96-well plate format with customized hardware is presented. Following protein precipitation of the plasma sample, an aliquot of the resulting supernatant is mixed with HMG-CoA reductase and 14C-labeled HMG-CoA prior to incubation. The product, 14C-mevalonic acid, is lactonized, separated from unreacted 14C-substrate, and counted in a liquid scintillation counter. Plasma HMG-CoA reductase inhibitor concentrations are measured against atorvastatin as the standard. Tecan Genesis 150 and 200 robotic workstations were used for the protein precipitation, enzyme incubation, and product separation. Results: The standard calibration range for the assay was 0.4–20 ng eq/mL. Intra-day precision (%CV) data for the calibration standard and quality control (QC) samples ( n = 5 replicates) were both ≤ 8%, with an accuracy between 88 and 113% of nominal values. Initial inter-day precision of the QC samples was ≤ 6%, with an accuracy range of 94–111% of nominal values. Discussion: The assay procedure provides high throughput analysis of clinical samples to support pharmacokinetic studies.