Mucociliary clearance (MCC) is the key defense mechanism in the upper airways, as the removal of debris-laden mucus in the sinuses completely depends on MCC. So far, how the nasal MCC is regulated remains unknown. Recently, mice deficient in genes encoding the components of MCC apparatus have been generated, which will allow investigators to conduct more in-depth nasal MCC studies. However, the methodology necessary to comprehensively evaluate the nasal MCC in this species is not well established. We therefore developed a novel method to measure nasal MCC in live mice using pinhole gamma camera. Insoluble radiolabeled particles were delivered into the noses of lightly anesthetized mice. The nasal clearance of these particles was measured continuously in a real-time manner. The effect of three different anesthetics-avertin, pentobarbital, and isoflurane-on nasal MCC was also determined. In mice anesthetized by 1.1% isoflurane, radiolabeled particles were immediately moved into the oropharynx, which was significantly accelerated by the treatment of hypertonic but not isotonic saline. According to the clearance rate, the mouse nasal MCC presented two distinct phases: a rapid phase and a slow phase. In addition, we found that isoflurane had a very small inhibitory effect on nasal MCC vs. both avertin and pentobarbital. This was further supported by its dose response. Collectively, we have developed a noninvasive method to monitor the real-time nasal MCC in live mice under physiological conditions. It provides more comprehensive evaluation on nasal MCC rather than assessing a single component of the MCC apparatus in isolation.