Tetrabenazine (TBZ) in rat plasma can be estimated using a reverse-phase high-performance liquid chromatography (RP-HPLC) approach employing benzoquinolizine as an internal reference. ICH M10 criteria were used to validate the created approach. Acetonitrile (ACN) and 0.1% formic acid were used in a ratio of 90:10 v/v for the separation, with a flow rate of 1 mL/min and a detection wavelength of 283 nm. Benzoquinolizine and TBZ had retention times (RT) of 3.665 and 5.064 minutes, respectively. The developed method's regression coefficient (r2) of 0.996 revealed that it is linear in the 50–250 ng/mL range. Indicating their accuracy and precision,