ET-26-HCl is a new analog of etomidate, a short-acting anesthetic drug, with less adrenal cortex inhibition. The pharmacokinetics of ET-26-HCl in rats needs to be determined for future clinical trials in human subjects. In order to facilitate the pharmacokinetic study, a liquid chromatography based tandem mass spectrometric (HPLC–MS/MS) method was developed and validated for quantification of ET-26-HCl and its major metabolite, ET-26-acid. These two compounds and gabapentin (internal standard) were extracted using a protein precipitation method with methanol and detected by Multiple Reaction Monitoring of m/z transition of 275.6–170.9, 217.7–113.1, and 172.5–154.3 for ET-26-HCl, ET-26-acid, and gabapentin respectively. This method was validated in terms of sensitivity, linearity, reproducibility, and stability. The HPLC–MS/MS method was found linear over the concentration ranges of 21.76–4352 ng/mL, and 18.62–3724 ng/mL with LLOQ of 21.76 and 18.62 ng/mL for ET-26-HCl and ET-26-acid respectively. The mean intra-day and inter-day accuracy was between 94.11–107.78%, while the precision was within the limit of 15.0% for all the quality control samples. A pharmacokinetic study was then conducted in rats following intravenous injection of 2.1, 4.2, and 8.4 mg/kg. The linear pharmacokinetics of ET-26-HCl was observed over the dose range of 2.1–8.4 mg/kg. The average terminal phase elimination half-lives were 0.87 and 1.03 h for ET-26-HCl and ET-26-acid respectively. In summary, an HPLC–MS/MS method for quantification of ET-26-HCl in rat plasma has been developed and successfully applied to a pharmacokinetic study.