Enzyme-inducing antiepileptic drugs (EIAEDs) are among the clinically most important inducers of cytochrome P450 (CYP) 3A4, but there is limited evidence regarding the comparative potency of each EIAED in raising CYP3A4 activity. The aim of this study was to estimate CYP3A4-inductive potency of EIAEDs by comparing CYP3A4 activity in patients treated with carbamazepine, phenobarbital, or phenytoin. Residual serum samples from patients treated with EIAEDs or levetiracetam were collected from a therapeutic drug monitoring service for analysis of 4β-hydroxycholesterol (4βOHC), which is an indicator of CYP3A4 activity. The samples were collected between January and September 2016 at Diakonhjemmet Hospital, Oslo, Norway. Concentration of 4βOHC, EIAEDs, and levetiracetam was measured by ultra-performance liquid chromatography tandem mass spectrometry. Kruskal-Wallis and Mann-Whitney tests were used for comparison of 4βOHC levels between the subgroups. In total, 4βOHC measurements for 343 and 339 patients treated with EIAEDs and levetiracetam, respectively, were included in the study. Compared with levetiracetam-treated patients, the median 4βOHC concentration was 3.3-fold, 5.8-fold, and 6.9-fold higher in patients using phenobarbital, phenytoin, or carbamazepine, respectively (P < 0.0001). Phenytoin users (n = 65) and carbamazepine users (n = 225) had 1.8- and 2.1-fold higher median 4βOHC concentration than phenobarbital users (n = 28), respectively (P ≤ 0.0001). This study shows that phenytoin and carbamazepine have approximately twice the CYP3A4-inducing potency of phenobarbital. The results indicate that 2-fold higher doses of CYP3A4-metabolized drugs may generally be required during concurrent treatment with phenytoin or carbamazepine compared with phenobarbital.