Accelerator mass spectrometry (AMS) is an ultra-sensitive analytical technique that has been used in the pharmaceutical setting for determining concentrations of drug-derived C-14 in biological samples after administration of nanocurie or low microcurie levels of C-14-labeled compounds. In recent years, AMS has been utilized to support absolute bioavailability (BA) studies where an intravenous (IV) [14C]microdose of drug is administered concurrently with an oral therapeutic dose. In this approach, plasma concentrations of unlabeled drug are quantitated with a validated LC-MS/MS method and [14C]drug concentrations are measured by AMS after chromatographic isolation. There are currently no universally accepted guidelines for AMS validation. Although accuracy and reproduciblity of AMS instrument measurements are considered to be good, quantitation is ultimately dependent upon sample processing. In particular, issues related to extraction recovery and verification of chromatographic separation of the parent compound from other drug-related components need to be addressed in order to obtain accurate drug measurement. A clinical absolute BA study of Onglyza™ (saxagliptin) was required for drug approval in some countries. This was the first time, to our knowledge, that an absolute BA study with the IV [14C]microdose was used in support of a regulatory approval. We therefore, elected to take a conservative approach for evaluating the AMS methodology, which included a validation and analysis of preclinical study samples prior to analysis of samples from the clinical study. The current investigation describes the evaluation of the methodology for measurement of [14C]saxaglitpin by AMS using a dog microtracer study. For the dog study, plasma samples were collected from 3 animals receiving an oral mictotracer dose of [14C]saxagliptin (0.071 mg/kg, 3.4 nCi/kg). At each time point, the samples from individual dogs were pooled, mixed well, and were then divided into 3 aliquots. The first aliquot was analyzed for saxagliptin using a validated LC-MS/MS assay. The remaining two aliquots were analyzed at two separate contract laboratories by AMS after chromatographic separation of saxagliptin from its metabolites. Standards and QC samples were also included in the AMS evaluation. Results indicated acceptable assay performance at both laboratories. Furthermore, plasma concentrations of saxagliptin in dog study samples determined by AMS were similar to those from the validated LC-MS/MS method and the values generated by the two AMS labs were comparable to each other.