Pulses of laser light have been employed, since as early as 1976, to produce intact gas phase peptide ions from solid samples. The resulting peptide ions could then be analyzed by mass spectrometry. These early investigations and subsequent measurements over the following decade produced useful mass spectra from only a few short peptides. In addition, the probability for obtaining a useful mass spectrum depended critically on the specific physical properties of the peptide (e.g., photoabsorption spectrum, volatility) under study. This situation changed dramatically, with the development by Karas and Hillenkamp. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) provides the means to volatilize proteins readily and to make the conditions for volatilization largely independent of the specific physical properties of the protein. This effect is achieved in two steps. The first step involves preparing an appropriate sample, by dilutely embedding proteins, in a matrix of small organic molecules that strongly absorb ultraviolet wavelength laser light. The second step involves ablation of bulk portions of this solid sample, by intense, short-duration pulses, of the laser light. In the ablation step, molecular components of the solid are put into the gas phase and ionized, producing intact protein ions. The molecular masses of these protein ions are easily determined by time-of-flight mass analysis. This chapter provides a practical guide to the application of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) for the analysis of peptides and proteins. It describes in detail the best methods that are currently available for preparing samples for MALDI-MS, because good sample preparation is the key to successful mass analysis. Aspects of the method that are important for obtaining high-quality data are discussed in this chapter. Finally, it describes a selection of strategies for studying proteins with this powerful new technique.