This is a case study of the difficulties caused by supersaturation of an analyte during the quantification of a drug in urine. To support the clinical development of the potent helicase–primase inhibitor ASP2151, we developed and validated simple and semi-automated methods for determining its concentrations in human plasma and urine. Samples were mixed with an internal standard labeled with stable isotopes, and then filtered. After filtration, the samples were injected into an online extraction, column-switching, liquid chromatography–tandem mass spectrometry analytical system using a validated method. Although quantifiable and reproducible plasma concentrations were obtained with clinical samples, we encountered difficulties with urine samples. Specifically, we found that urinary drug concentrations in some samples ranged between 5 and 67.1 μg mL−1, and exceeded the aqueous saturation concentration (5 μg mL−1), at which dilution integrity was confirmed. In a follow-up experiment using spiked samples, the urine method failed to accurately quantitate ASP2151 concentrations at 10, 25, and 50 µg mL−1 (relative error: −23.5% to −17.2%, coefficient of variation: ≤7.9%). In vitro experiments revealed that urine samples at high ASP2151 concentrations became heterogeneous during sample handling and storage. The problem was solved by revising the sample collection and dilution methods which were subsequently successfully applied to a clinical study.