Un-differentiated (UD) and well-differentiated (WD) normal human primary bronchial/tracheal epithelial cells are important respiratory cell models. Mature, WD cells which can be derived by culturing UD cells at an air-liquid interface represent a good surrogate for in vivo human airway epithelium. The overall protein profile of WD cells is poorly understood; therefore, the current study evaluated the proteomic characteristics of WD and UD cells using label-free LC-MS/MS and LC-PRM/MS. A total of 3,579 proteins were identified in WD and UD cells. Of these, 198 proteins were identified as differentially expressed, with 121 proteins upregulated and 77 proteins downregulated in WD cells compared with UD cells. Differentially expressed proteins were mostly enriched in categories related to epithelial structure formation, cell cycle, and immunity. Fifteen KEGG pathways and protein interaction networks were enriched and identified. The current study provides a global protein profile of WD cells, and contributes to understanding the function of human airway epithelium.