<p>Furosemide is a diuretic drug used to increase urine flow in order to reduce the amount of salt and water in the body. It is commonly utilized to treat preterm infants with chronic lung disease of prematurity. There is a need for a simple and reliable quantitation of furosemide in human urine. We have developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry method for furosemide quantitation in human urine with an assay range of 0.100-50.0&nbsp;μg/ml. Sample preparation involved solid-phase extraction with 10&nbsp;μl of urine. Intra-day accuracies and precisions for the quality control samples were 94.5-106 and 1.86-10.2%, respectively, while inter-day accuracies and precision were 99.2-102 and 3.38-7.41%, respectively. Recovery for furosemide had an average of 23.8%, with an average matrix effect of 101%. Furosemide was stable in human urine under the assay conditions. Stability for furosemide was shown at 1&nbsp;week (room temperature, 4, -20 and -78°C), 6&nbsp;months (-78°C), and through three freeze-thaw cycles. This robust assay demonstrates accurate and precise quantitation of furosemide in a small volume (10&nbsp;μl) of human urine. It is currently being implemented in an ongoing pediatric clinical study.</p>