Analytical methods have been developed for the detection and quantification of cyclosporin A (CsA) in blood using time-of-flight secondary-ion mass spectrometry (TOF-SIMS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). Linear calibration curves were obtained for both methods in the range 25-2000 ng/mL in whole blood. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 7 and 23 ng/mL, respectively, for the TOF-SIMS method; the LOD and LOQ were 10 and 33 ng/mL, respectively, for the MALDI/TOF-MS method. Coefficients of variation ranged from 3 to 5% for the TOF-SIMS method and from 4 to 8% for the MALDI/TOF-MS method. Blood samples were also analyzed by a nonspecific method, fluorescence polarization immunoassay (FPIA), and by a specific method, high-performance liquid chromatography (HPLC). The TOF-MS results are in good agreement with the HPLC results, but not with the FPIA results. The TOF-MS methods can also provide information about CsA metabolites.