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Objectives The objective of the current investigation was to develop a simple, rapid method for determining in-vivo 5-hydroxytryptamine type 4 receptor ( 5-HT4R) occupancy in rat brain using non-radiolabelled SB207145 as a tracer for accelerating the drug discovery process. Methods In-vivo tracer optimization studies for tracer dose, survival intervals and brain distribution profile were carried out in rats. The tracer was pharmacologically validated using potent well-characterized 5-HT4R ligands. The brain regional concentrations of tracer ( SB207145); plasma and brain concentrations of 5-HT4R ligands were quantified using high-performance liquid chromatography coupled with a tandem mass spectrometric detector ( LC-MS/ MS). Key findings SB207145 showed a higher specific binding in striatum (1.96 ng/g) and lower binding in cerebellum (0.66 ng/g), which is consistent with findings of other published 5-HT4R expression studies. Pretreatment with potent 5-HT4 ligands dose-dependently reduced striatal SB207145 concentration and the effective dose to achieve 50% receptor occupancy ( ED50) values were 4.8, 2.0, 7.4, 9.9, 3.8 and 0.02 mg/kg for GR113808, piboserod, prucalopride, RS67333, TD8954 and PF04995274, respectively. Conclusions Results from the mass spectrometry approach to determine 5-HT4R occupancy in rat brain are comparable with those reported using radiolabelled scintillation spectroscopy methods. In conclusion, the LC-MS/ MS characterization permits use of tracer at a preclinical stage in high-throughput fashion as well as characterization of target expression. [ABSTRACT FROM AUTHOR]