Isavuconazole (ISZ) is used in the treatment of aspergillosis and mucormycosis. The purpose of this study was to evaluate the therapeutic drug monitoring (TDM) of ISZ samples from a clinical setting performed at Statens Serum Institut. Isavuconazole serum concentrations were determined by fluorescent detection on a UHPLC. Serum-ISZ (s-ISZ) results were included and compared to those of serum-voriconazole (s-VRZ) in a 33 month period from March 2017. Clinical data were obtained for patients receiving ISZ. The therapeutic range was initially 2-10 mg/L, but was adjusted to 2-5 mg/L during the study period except for selected patients with Mucorales infections who received off-label doses of ISZ. A total of 273 s-ISZ and 1242 s-VRZ measurements from 35 and 283 patients, respectively, were included. Seventeen patients had received both ISZ and VRZ with TDM within the study period. The median s-ISZ was 4.3 mg/L (0.5-15.4 mg/L) with 83% of measurements within the therapeutic index. The median s-VRZ was 2.6 mg/L (0.2-21.9 mg/L) with 67% of measurements within the therapeutic index. The median intra-/interindividual coefficient of variation (CV) was 43.4%/54.8% for ISZ compared to 53.2%/83.3% for VRZ. For patients receiving ISZ, the adverse events were mostly gastroenteric and few drug-drug interactions were observed. Furthermore, immediate change from ISZ to VRZ treatment seemed to lead to prolonged metabolism of ISZ with detection up to 35 days after discontinuation. The majority of patients achieved s-ISZ levels well within the therapeutic range with less intra/interindividual CV than patients receiving VRZ.