An analytical method based on high-performance liquid chromatography coupled to quadrupole tandem mass spectrometry was developed for the quantitative determination of four phosphatidylcholines (PCs) in human exhaled breath particles. Analytes were conveniently collected on an electrostatic polymer filter and extracted with methanol prior to analysis. Chromatographic separation was performed on an ultraperformance liquid chromatographic ethylene bridged hybrid phenyl column using a mobile phase consisting of water and methanol containing 4 mM ammonium formate and 0.1% ammonia. The mass spectrometer operated in positive electrospray ionization and selected reaction monitoring mode. Detection limits for PC 16:0/16:0 (dipalmitoylphosphatidylcholine, DPPC), PC 16:0/18:1, PC 16:0/18:2, and PC 18:0/18:2 were <0.01 ng/filter. Method recoveries at concentration levels of 0.1 and 10 ng/filter were 100-110% and 101-121%, respectively. Acceptable precision with coefficients of variation <20% and accuracies of 100% ± 20% were achieved. Identification of the individual PCs was performed on the basis of two product ions with correct ion ratios and chromatographic retention times. The highest amount in exhaled breath was found for DPPC with median concentration 1.14 ng/filter (range 0.6-21 ng/filter), and median molar ratios of DPPC/PC (16:0/18:1) of 1.98 (range 0.48-2.75). A different pattern with lower molar ratio (∼0.15) was found for oral fluid. The most significant element of this study was to use a precolumn in the LC system and to collecting exhaled particles in an electret polymer filter. Due to chromatographic interference by background contamination, an isolator column (PFC kit) was installed in between eluent mixer and injector to reduce contamination. This is the first LC/MS study where the method was successfully applied to analyze PCs in human exhaled breath by using a simple and convenient collection procedure.