Cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ion channel in the apical plasma membrane of epithelial cells. Mutations in the gene coding for CFTR cause cystic fibrosis (CF). A major cellular dysfunction is insufficient apical plasma membrane expression of the protein. Its correction is important for developing new CF therapeutics and treatments, which requires a sensitive and precise method for quantifying apical plasma membrane CFTR. We report the first method of liquid chromatography−tandem mass spectrometry for quantifying endogenous and overexpressed CFTR in HT29 and BHK cells. For low level of endogenous CFTR from HT29, the target protein in the cell lysate was enriched by immunoprecipitation using anti-CFTR antibody MAB3484 or M3A7. For overexpressed CFTR from BHK, the cell lysate prepared by differential detergent fractionation or surface biotinylation was used directly without immunoprecipitation. Proteins in the enriched CFTR preparations or cell lysates were digested with proteases, and a surrogate marker peptide designated as CFTR01 (NSILTETLHR) was successfully quantified using the method of multiple reaction monitoring and stable isotope dilution with an 18O-labeled reference peptide (CFTR01-18O4) as the internal standard. CFTR quantified in this work ranged from a few tens of picograms to low nanograms per million of cells.