Internal standards were used to demonstrate that matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be applied to the quantitative analysis of low molecular weight polar compounds. Three examples were tested: a standard curve for 3,4-dihydroxyphenylalanine (DOPA) was prepared using a stable isotope analogue (i.e., [13C6]DOPA) as an internal standard; [2H16]-acetylcholine was employed as an internal standard for the quantification of acetylcholine; and in the final example, the peptide Ac-Ser-Ile-Arg-His-Tyr-NH2 was used as an internal standard for the quantification of the peptide H-Ser-Ala-Leu-Arg-His-Tyr-NH2. In each instance, straight line fits (r2 > 0.95) demonstrate that MALDI is a viable approach for the quantitative analysis of low molecular weight analytes.