This study describes a matrix-assisted laser-desorption-ionization (MALDI) mass spectrometry for rapid screening of 12 diuretics in spiked urine. C60 is used as the matrix for MALDI. Diuretics are directly analyzed by C60-MALDI without previous derivatization. The fact that most diuretic molecules contain sulfate groups accounts for why anions of the molecules can be easily desorbed and ionized in MALDI. Using C60 as the matrix is advantageous because of the low background in the low mass range on the negative MALDI mass spectrum. A clear mass window between m/z 200 and 600 in negative ion mode is also obtained. Only a minimum amount of the sample (< 1 μL) is necessary to perform the analysis. The detection limit of diuretics is approximately 0.1-1 μg/mL.