Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. D-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of D-amphetamine in drug research and its interest in toxicologic-forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of D-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of D-amphetamine in rat blood using MS(3) scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC-MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1mm x 30mm, 3.5microm) using gradient elution at a flow rate of 1.0mL/min over a 2min run time. An Applied Biosystems API4000 QTRAP mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS(3) scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1-->119.1-->91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1-->208.2. The linear dynamic range was established over the concentration range 0.5-1000ng/mL (r(2)=0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS(3)) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.