The data in this study support the use of MS coupled to integrated microscale LC to match the sensitivity of ligand binding assays while minimizing the sample volume required. Although there are specific platforms that allow for LBA analysis from as little as 4–8 μl of sample, many LBA analyses require more sample and all are still subject to the acknowledged shortcomings of LBAs as a general technique. Cumulative sensitivity gains of 15–30-fold were realized when comparing final results from 2.1 mm ID scale LC–MS methods to those obtained on a 150 μm ID integrated microfluidic device LC–MS system. The data acquired using this platform easily met recommended accuracy and precision guidelines and analysis speed (10–12 min run times) was adequate for high throughput bioanalytical assays. Use of integrated microscale LC shows promise for quantification of peptides from sample volumes as low as 25 μl, facilitating single animal PK models and biomarker discovery research.