In recent years, it has become clear that the presence of high concentrations of 1-OH midazolam glucuronide is probably the cause of unexplained prolonged midazolam comas in patients with poor renal function. Until recently, only indirect methods for the analysis of this glucuronide were known, which had several disadvantages, such as a long analysis period (>6 hours). This article describes the validation of a method for the direct analysis of this compound in human serum, using reversed-phase ion-pair high-performance liquid chromatography (HPLC) in combination with solid phase extraction. The intraday and interday coefficients of variation have values below 6% for different possible serum concentrations. The limit of quantification (0.1 mg/L) is much lower than concentrations found in patients with a coma caused by the accumulation of 1-OH midazolam glucuronide. Recovery of 1-OH midazolam glucuronide is almost 100% at three different serum concentrations. Linearity is confirmed for normal serum levels (<1 mg/L) and for serum levels that might occur in patients with impaired renal function (<20 mg/L). Detection is performed at 254 nm with a diode array detector, which can also be used to check the peak purity in case of unexpected impurities.