Although levetiracetam presents an easy dosing and tolerability, therapeutic drug monitoring may be recommended in certain situations. Measurement of levetiracetam in serum plasma is commonly done by high performance liquid chromatography (HPLC). After ARK Diagnostics marketed an enzyme immunoassay (IA) for levetiracetam in serum or plasma, automated determinations are possible. In this study, the performance of this immunoassay and the impact of automation on the follow-up in patients treated with levetiracetam is evaluated. We also detected those subpopulations of patients who may benefit the most from this therapeutic drug monitoring. Samples from 50 outpatients diagnosed with epilepsy and treated with levetiracetam were collected. This new IA was performed on the Architect c4000 analyser and compared with the HPLC. Then, a retrospective observational study that included serum samples of levetiracetam for 24 months, was conducted to evaluate the impact of automattion and the influence of some variables (age, sex, renal function, and co-administration of valproic acid and glucuronidation-inducing drugs) in levetiracetam apparent oral clearance (CLp/F) by a multivariate linear regression. The mean high-performance liquid chromatography quantified concentration (CpHPLC) was 18.43 mcg/mL (95% CI: 15.48 to 21.39) and immunoassay concentration (CpEI) was 18.35 mcg/mL (95% CI: 15.20 to 21.50) (P=0.861). The Pearson's linear correlation coefficient obtained in the analysis was r2=0.88, according to the following equation: CpHPLC=-0.29+1.01 CpEI. The intraclass correlation coefficient was 0.95 (95% CI: 0.91 to 0.97). After IA implementation, the number of levetiracetam determinations increased in 76.27%. The median of Clp/F was higher (P<0.001) in inducers (4.36 L/h; IQR:3.29-5.44) and lower (P<0.001) in glomerular filtration rate (GFR) <60 mL/min (2.7 L/h; IQR: 0.58-3.85). The Ark method performed on the Architect is fully acceptable and can be used routinely to measure levetiracetam plasmatic concentration levels. It has demonstrated the need for closer monitoring in patients with renal failure or co-administration of glucuronidation-inducing drugs.