Four 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) derivatives with molecular weights of 339-1131 Da were synthesized to investigate their ionization behaviors in fast atom bombardment (FAB), electron ionization (EI), direct analysis in real time (DART), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). These include 4-dodecanoylamino-2,2,6,6-tetramethyl-1-piperidinyloxy (1), 4-dodecylamino-2,2,6,6-tetramethyl-1-piperidinyloxy (2), N,N'-bis(2,2,6,6-tetramethyl-1-oxyl-4-piperidinyl)dodecanediamide (3), and bis-cholesterol TEMPO derivative 4. For TEMPO derivatives 1 and 2 (molecular weights: 353 Da and 339 Da), [M]