Salsolinol (Sal) and N-methylsalsolinol (NMSal) are endogenous neurotoxins in the brain, which are the key causative agents of the death of dopaminergic neurons. Due to their strong polarity, they have weak retention and are difficult to quantify using the C₁₈ column. In the present study, a sensitive and selective LC/MS method for the determination of Sal and NMSal was developed and validated using a novel pentafluorophenylpropyl column performed on high performance liquid chromatography electrospray ionisation triple quadrupole. The calibration curve was linear over the range of 0.098–25 nM and 3.91–250 nM for Sal and NMSal, respectively. The lower limit of quantitation is 0.098 nM and 3.91 nM for Sal and NMSal. The inter-day and intra-day precision and accuracy for all samples were acceptable. Furthermore, the assay has also been applied for the determination of Sal and NMSal in rat brain.