While mortality rates from heart disease and cancer have seen a steady decline, chronic pulmonary disease mortality remains steady with projections estimating chronic lung conditions to become the number one cause of death worldwide by 2030. The respiratory tract is composed of a large epithelial surface that gets exposed to a variety of pathogens and pollutants inhaled with every breath. To protect against these constant insults, a key mechanism that has evolved in vertebrates is the mucociliary transport of the tracheobronchial tree. This system comprised of the trachea, bronchi, and bronchioles is lined by multiple motile cilia that beat in a metachronal, synchronous wave to generate cephalad flow of mucus away from lung alveoli. The importance of this mechanism becomes evident in patients suffering from primary ciliary dyskinesia, a congenital recessive autosomal disorder characterized by mutations in key ciliary proteins leading to immotile or dyssynchronous respiratory cilia. This ciliary dysfunction in turn leads to recurrent lung infections and bronchiectasis, ultimately necessitating lung transplantation. In our world of ever-increasing air pollution, novel viral pathogens, and multidrug-resistant bacteria, the importance of adequate mucociliary clearance cannot be overstated. In this Methods Collection, we will showcase articles from leading authorities on the assessment of ex vivo, in vivo, and patient-centered methods for the evaluation of mucociliary clearance. Our hope is that the reader will find it useful for generating data in their own basic and clinical research endeavors to identify targets for novel therapies and drug development for a host of pulmonary pathologies expected to only grow and increase in importance over time.