We developed and validated a reverse-phase high performance liquid chromatography (RP-HPLC) method for the purity assay of BZM. BZM is a precursor and free ligand of [123I]IBZM (iodobenzamide) that is a SPECT imaging agent for striatal dopaminergic D2/D3 receptor (D2R). The chromatographic separation was achieved on a Zorbax Eclipse XDB-C18 column with a gradient mobile phase consisting of 10 mM ammonium acetate buffer, pH 7.0 and acetonitrile at a flow rate of 0.5 mL/min. The absorbance at 254 nm against the concentration of BZM over the range 0.5-5.5 μg was linear, with a correlation coefficient above 0.9997. The limit of detection (LOD) and quantification (LOQ) of major impurity (impurity A, tR = 3.85 min) in BZM were 0.028% and 0.094%, respectively. Method validation parameters, including specificity, precision, accuracy, linearity, LOD/LOQ, robustness and solution stability were evaluated, indicating the potential of this method in pharmaceutical quality control. Moreover, the most sensitive precursor to product ion transitions for the identification of BZM and ‘cold’ IBZM by LC-ESI-MS/MS were found to be m/z 279.0-112.0 and 405.0-112.0, respectively. The present results allow identification of fragmentation ions and proposition of the pathways of BZM and ‘cold’ IBZM, showing that the feasibility of this method for quantification of free ligand BZM in SPECT imaging agent [123I]IBZM injection.