Therapeutic drug monitoring (TDM) of voriconazole is recommended for personalizing doses. The objective of this study was to compare the enzyme immunoassay developed by ARKTM Diagnostics Inc. for the quantification of voriconazole adapted to the Architect C4000 autoanalyzer (Abbott®) with ultra-performance liquid chromatography using ultraviolet detector (UPLC-UV) method. Linearity, precision and accuracy of both methods were validated according to the Food and Drug Administration (FDA) and European Medicines Agency guidelines. The limit of quantification (LOQ) of the UPLC-UV method was determined experimentally. Both methods were applied to the analysis of 62 samples from patients. Correlation was evaluated by Passing-Bablok analysis and the concordance by the Bland-Altman method. Dosage recommendations were generated; the discordances according to the technique were evaluated. All validation parameters determined for UPLC-UV met the criteria set out and LOQ of 0.1 μg/mL was established. However, when the enzyme immunoassay was used to determine concentrations ≤1 μg/ml, CVs were >20%. A linear correlation between both methods was found. However, an overestimation of immunoassay (systematic error of 0.39 μg/mL) was detected. In 11.3% of the samples, the differences in concentrations when they were determined by different techniques would imply a different therapeutic regime. These samples had concentrations close to 1 μg/mL. Although both techniques can be used for TDM of voriconazole, when a value close to the lower limit of the therapeutic range is determined by the ARKTM immunoassay, it would be better to verify the result by a non-automated technique to avoid possible underdosing.