MTBT 1466A is a monoclonal antibody designed to bind to mature human TGFβ3 in human tissue and systemic circulation. To evaluate binding of this therapeutic, a mature TGFβ3 assay was needed to be able to monitor pharmacodynamic responses in non-human primate (NHP) studies. However, mature TGFβ3 levels in systemic circulation are very low and require development of a highly sensitive assay for detection. This study describes the development of a highly sensitive, drug-tolerant pharmacodynamic biomarker assay for demonstrating target engagement in a pre-clinical study using MTBT1466A. Since mature TGFβ3 is a dimer, a single MAb was used as both the capture and detection antibodies. This assay was developed on the SMCxPRO platform and qualified based on current accepted criteria for biomarker assays. The assay demonstrated specificity to mature TGFβ3, with a lower limit of quantification of 31.3pg/mL. Although baseline levels of mature TGFβ3 were below the assay detection limit in 40% of animals within our study, 2- to 16-fold increases were observed in many of the animals following multiple-dosing regimen.