© 2015 ARS-AAOA, LLC. Background: Measuring ciliary beat frequency (CBF) is a technical challenge and difficult to perform in vivo. Doppler optical coherence tomography (D-OCT) is a mesoscopic noncontact imaging modality that provides high-resolution tomographic images and detects micromotion simultaneously in living tissues. In this work we used D-OCT to measure CBF in ex vivo tissue as the first step toward translating this technology to clinical use. Methods: Fresh ex vivo samples of rabbit tracheal mucosa were imaged using both D-OCT and phase-contrast microscopy (n = 5). The D-OCT system was designed and built to specification in our lab (1310-nm swept source vertical-cavity surface-emitting laser [VCSEL], 6-μm axial resolution). The samples were placed in culture and incubated at 37°C. A fast Fourier transform was performed on the D-OCT signal recorded on the surface of the samples to gauge CBF. High-speed digital video of the epithelium recorded via phase-contrast microscopy was analyzed to confirm the CBF measurements. Results: The D-OCT system detected Doppler signal at the epithelial layer of ex vivo rabbit tracheal samples suggestive of ciliary motion. CBF was measured at 9.36 ± 1.22 Hz using D-OCT and 9.08 ± 0.48 Hz using phase-contrast microscopy. No significant differences were found between the 2 methods (p > 0.05). Conclusion: D-OCT allows for the quantitative measurement of CBF without the need to resolve individual cilia. Furthermore, D-OCT technology can be incorporated into endoscopic platforms that allow clinicians to readily measure CBF in the office and provide a direct measurement of mucosal health.