A robust high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3′,5′-cyclic adenosine-13C5 monophosphate (13C5-cAMP) and 3′,5′-cyclic guanosine-13C,15N2 monophosphate (13C15N2-cGMP) were used as surrogate analytes to measure endogenous 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4 M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC–MS/MS. The calibration curves for both analytes ranged from 5 to 2000 ng/g and showed excellent linearity (r2 > 0.996). Relative surrogate analyte-to-analyte LC–MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13C5-cAMP and 13C15N2-cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13C5-cAMP and 13C15N2-cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies.