Airway epithelial cells play an important role in the pathogenesis of inflammatory lung diseases such as asthma, cystic fibrosis and COPD. Studies concerning the function of the lipid metabolism of the airway epithelium are so far based only on the detection of lipids by immunohistochemistry but quantitative analyses have not been performed. Although recent advances in mass spectrometry have allowed to identify a variety of lipid classes simultaneously in isolated tissue samples, up until now, these methods were not suitable to analyze lipids in the airway epithelium. To determine all major lipid classes in airway epithelial cells, we used an LC–MS-based approach that can easily be combined with the specific isolation procedure to obtain epithelial cells. We tested the suitability of this method with a mouse model of experimental asthma. In response to allergen challenge, perturbations in the sphingolipids were detected, which led to increased levels of ceramides. We expanded the scope of this approach analysing human bronchus samples without pathological findings of adenocarcinoma patients. For the human lung epithelium an unusual lipid class distribution was found in which ceramide was the predominant sphingolipid. In summary, we show that disease progression and lipid metabolism perturbation can be monitored in animal models and that the method can be used for the analysis of clinical samples.