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Introduction: Conventionally, receptor occupancy assays employ radiolabelled tracer. However, recent advances with non-radiolabelled tracers brought a revolution in target engagement assays. Non-radiolabelled tracer based receptor occupancy uses LC-MS/MS based quantification. It offers simultaneous quantification of more than one tracer; thus, provides the feasibility of evaluating multiple targets in a single animal. In the present study, we demonstrated simultaneous measurement of serotonin 1A, serotonin 2A, and dopamine 2 receptor occupancy using non-radiolabelled tracers in rats., Method: Non-radiolabelled WAY-100635 or MDL-100,907 or raclopride were used as tracers for 5-HT 1A , 5-HT 2A , and D 2 receptors, respectively. In-vivo brain distribution of these tracers was measured after administration as individual or as a mixture of tracers (cocktail tracer). Similarly, in-vitro brain free fractions were evaluated with the single and cocktail tracer in brain homogenates. The mass spectrometer was used for simultaneous quantification of tracers in both in-vivo and in-vitro samples. A ratio method was employed for calculation of receptor occupancy after single and cocktail tracer administration. Pindolol, olanzapine, and ziprasidone were used as tool compounds for demonstrating receptor occupancy at 5-HT 1A , 5-HT 2A , and D 2 receptors., Result: In optimization studies, regional distribution and concentration ratios (specific to non-specific) of these tracers were unaltered with individual and cocktail tracer. Non-significant variability was observed in brain free fraction of tracers' indicating the minimal binding interactions in this tracer combination. The half-maximal effective dose (ED 50 ) for pindolol (5-HT 1A 1.37 & 2.42mg/kg, i.v.), olanzapine (5-HT 2A 1.37 & 2.12 and D 2 1.90 & 2.72mg/kg, p.o.), and ziprasidone (5-HT 1A 10.92 & 9.57; 5-HT 2A 0.03 & 0.04 and D 2 0.11 & 0.08mg/kg, i.v.) were comparable with individual or cocktail tracer., Discussion: The present study demonstrated the utility of non-radiolabelled tracers in simultaneous measurement of multiple target engagement. Use of this method will minimize the time, in addition to the cost in translational research., (Copyright © 2017 Elsevier Inc. All rights reserved.)