A new tandem time-of-flight (TOF-TOF) instrument has been developed by modifying a standard matrix-assisted laser desorption ionization (MALDI)-TOF instrument to make high-performance, high-energy collision-induced dissociation (CID) MALDI tandem mass spectrometry (MS) a practical reality. To optimize fragment spectra quality, the selected precursor ion is decelerated before entering a floating collision cell and the potential difference between the source and the collision cell defines the collision energy of the ions. Standard operating conditions for tandem MS use a 1-kV collision energy with single-collision conditions and increased laser power for ion formation. Hence, both high- and low-energy fragments are observed in MALDI TOF-TOF spectra. On standard peptides, sensitivities down to 1 fmol are demonstrated. On a mixture of two solution tryptic digests at the 25-fmol level, 23 spectra were sufficient to result in proper database identification.