The characterization of absolute bioavailability (BA) is useful for non-intravenous (iv.) formulations during drug development and is required by some health authorities. A study design of co-administrating an iv. isotopically labeled microdose with a therapeutic oral dose is a viable approach for the determination of human PK and has been accepted by regulatory agencies. The implementation of an iv.-microdose with oral therapeutic dose in absolute BA studies speeds up clinical development. In recent years, AMS to measure a radiolabeled microdose has been utilized to support several clinical absolute BA studies. An alternative approach for conducting microdose studies is using LC-MS/MS alone to quantitate both the iv. drug and the oral drug. Because both labeled and unlabeled drugs can be measured simultaneously with LC-MS/MS, it is cost effective. However, for compounds with high volume of distribution and/or poor LC-MS/MS response, AMS still provides a superior LLOQ. In this Perspective, we discuss a paradigm for selecting either an LC-MS/MS or AMS-based approach for generating concentration data in absolute BA studies dependent on the required sensitivity.