In the present study the bioavailability and pharmacokinetics properties of pantoprazole (proton pump inhibitor)/amitriptyline (tricyclic antidepressant) in novel formulated effervescent granules was estimated in rabbit plasma using a validated, selective and rapid LC-MS/MS method. Separation and detection of pantoprazole, amitriptyline and internal standards namely omeprazole and dothiepin, respectively, were achieved at ambient column temperature on C
18
. Acetonitrile: 4mM ammonium acetate solution (comprising 0.05 % formic acid) (40:60, v/v) was used as mobile phase and the flow rate of 0.6 mLmin
-1
was applied. Liquid-liquid extraction technique with diethyl ether: dichloromethane (70:30, v/v) was used to extract the cited drugs from rabbit plasma. Multiple reactions monitoring (MRM) in the positive ionization mode was carried out for quantification. The method was validated over linear concentration range of 0.01-4μgmL
−1
and 0.001–0.1 μgmL
−1
for Pan and Ami respectively, with regression coefficient (r
2
) ≥ 0.9961. The intra- and inter-run precisions (%CV) were ≤4.03. The extraction recoveries were in the range of 95.92%–100.24 %. Pan and Ami were stable during three freeze-thaw cycle and post-preparative stability. The work also aimed to formulate immediate release novel effervescent granules by melt granulation technique. Nine formulae were assessed by validated dissolution test for their micrometric properties and dissolution profile. Experimental design was applied to select formula that fulfilled the desired criteria of optimum release of pantoprazole and amitriptyline with optimum micrometric properties for the study. A single period randomized open-label parallel design was applied on Chancellor's rabbit. The selected formula showed superior pharmacokinetic parameters for pantoprazole and amitriptyline than that of marketed products.