QX-OH, a new synthetic local anesthetic, produced concentration-dependent, reversible, and long-acting local anesthesia in animal models, with moderate local toxicity. As part of preclinical research for drug development, we developed and validated a method for the determination of QX-OH in the plasma, muscle, and sciatic nerve using liquid chromatography–mass spectrometry. After a simple protein precipitation procedure, analysis was performed on an Extend C18 column (100 mm × 3 mm, 3.5 μm) by isocratic elution with 0.05% formic acid/acetonitrile (78:22, v/v) at a flow rate of 0.3 mL/min. A multiple-reaction monitoring mode at the transitions of m/z 279.1 → 102.1 for QX-OH and m/z 275.2 → 126.1 for an internal standard (ropivacaine hydrochloride) was used for the quantification, with a positive electrospray ionization interface. The approach was validated as per the U.S. Food and Drug Administration guidelines and successfully used in a pharmacokinetic study of QX-OH after a sciatic nerve block with 0.2 mL of 35 mM QX-OH. The results demonstrated that the new local anesthetic, QX-OH, had a high concentration in tissue, low systemic exposure, and long duration in the sciatic nerve.