Nitrofurantoin is an antimicrobial drug that has been used in the treatment of lower urinary tract infections for more than 50 years. Despite its long use, surprisingly little is known of the pharmacokinetics of nitrofurantoin, whereas this is essential to optimize patient treatment. We developed a novel analytical method for the quantification of nitrofurantoin in plasma and urine using ultra-high performance liquid chromatography and diode array detection to allow pharmacokinetic studies in these two matrices. The sample preparation method consisted of protein precipitation for plasma and liquid-liquid extraction for urine. 100 μL was needed for the sample preparation. Furazolidone was used as internal standard. Gradient chromatographic separation was performed on a HSS-T3 column. UV detection was performed at a wavelength of 369 nm. The analysis time was 5 min. The method was successfully validated according to the FDA-guidelines (2018). Linearity was confirmed over a concentration range from 50 to 1250 μg/L in plasma and from 4 to 200 mg/L in urine (r2 > 0.95). Validation results of five QC concentrations for plasma and urine, respectively, are for within-day accuracy <± 13% in both matrices, for between-day accuracy <± 7% and <± 9%, for within-day precision <10% and <4% and for between-day precision <10% and <5%. Plasma samples are stable for seven days at 4 °C, and for 2 years at -20 °C and-80 °C. Urine samples are stable for at least seven days at 4 °C and at room temperature and for 2 years at -20 °C andat -80 °C, except from the lower concentrated samples, which are only stable at -80 °C. All samples were kept from daylight using amber colored glassware. The presented method meets all validation requirements and was successfully used in a clinical study where the pharmacokinetics of nitrofurantoin were investigated in healthy volunteers. The easy sample preparation method and the short analysis time make this method suitable for use during routine clinical practice to study the pharmacokinetics of nitrofurantoin.