Mucociliary clearance has long been known to be a significant innate defence mechanism against inhaled microbes and irritants. Important knowledge has been gathered regarding the anatomy and physiology of this system, and in recent years, extensive studies of the pathophysiology related to lung diseases characterized by defective mucus clearance have resulted in a variety of therapies, which might be able to enhance clearance from the lungs. In addition, ways to study in vivo mucociliary clearance in humans have been developed. This can be used as a means to assess the effect of different pharmacological interventions on clearance rate, to study the importance of defective mucus clearance in different lung diseases or as a diagnostic tool in the work-up of patients with recurrent airway diseases. The aim of this review is to provide an overview of the anatomy, physiology, pathophysiology, and clinical aspects of mucociliary clearance and to present a clinically applicable test that can be used for in vivo assessment of mucociliary clearance in patients. In addition, the reader will be presented with a protocol for this test, which has been validated and used as a diagnostic routine tool in the work-up of patients suspected for primary ciliary dyskinesia at Rigshospitalet, Denmark for over a decade.