Hydroxyurea (HU) has been used in the treatment of chronic myeloid leukaemia (CML) and other myeloproliferative malignancies. Considering patient's wide variation in clinical response to HU, a new and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated to monitor patients' compliance to treatment and investigate the pharmacokinetics of HU in patients with CML. Stable isotope labeled HU-13C1,15N2 was used as internal standard. Plasma samples were treated with acetonitrile to precipitate protein. The supernatant was injected directly without derivatization and separated on a hydrophilic interaction liquid chromatography column. HU was quantitatively analyzed with a mobile phase of acetonitrile-1.5 mM ammonium formate (90:10, V:V) within 3 min. The proposed method provided a linearity range of 1–200 μg/mL. The coefficients of variation for intra- and inter-day precision were less than 2.07% and 4.28%, respectively, while the accuracy (bias) was in the range of −3.77 to 2.96%. This method was satisfactorily applied to the determination of HU in two patients with CML. It is suitable for supporting pharmacokinetic studies and clinical therapeutic monitoring.