An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS). The plasma samples were prepared by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide column (2.1 mm × 50 mm, 1.7 μm) with a 3 min gradient elution at a flow rate of 0.5 mL/min. For mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were m/z 144 → 98 and m/z 144 → 56 for cyclocreatine and m/z 148 → 102 for the internal standard (D4-cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were constructed in the range of 0.01–25 μM. The correlation coefficient of the calibration curves was greater than 0.99. The mean intraday assay accuracy for all quality control (QC) replicates was between 93 and 105%. The mean intraday assay precision (CV%) was 1.9-11% for all QC levels. The HILIC–UPLC–MS/MS method was successfully applied in pharmacokinetic (PK) studies of cyclocreatine in mice and rats for the first time. After a single 30 mg/kg oral administration in mice and rats, the AUC0-∞ (area under the curve) was 84.1 μg h/mL and 91.7 ± 18.0 μg h/mL, respectively.