Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is one of the most important techniques for chemical imaging of nanomaterials and biological samples with high lateral resolution. However, low ionization efficiency limits the detection of many molecules at low concentrations or in very small volumes. One promising approach to increasing the sensitivity of the technique is by the addition of a matrix that promotes ionization and desorption of important analyte molecules. This approach is known as matrix-enhanced secondary-ion mass spectrometry (ME-SIMS). We have investigated the effect of matrix acidity on molecular ion formation in three different biomolecules. A series of cinnamic acid based matrixes that vary in acidity was employed to systematically investigate the influence of matrix acidity on analyte ion formation. The positive ion signal for all three biomolecules showed a strong increase for more acidic matrixes. The most acidic matrix was then vapor-deposited onto mouse brain sections. This led to significant enhancement of lipid signals from the brain. This work indicates that proton donation plays an important role in the formation of molecular ions in ME-SIMS.