A comprehensive study investigated the effect of polar protic (methanol and water) and polar aprotic (acetonitrile and acetone) solvents on the chromatographic separation and negative-ion electrospray (ESI) response of 49 diverse small, acidic molecules. Flow injection experiments on a triple quadrupole were used to measure the response in neat solvents after optimization of source conditions and implementation of a rigorous quality control program (the later ensured that changes in analyte response were due to the analyte/solvent measured and not changes in instrument performance over time). In all solvents, compounds with electron-withdrawing groups and extended conjugation ionized best due to resonance and inductive effects. Ionization was greatest in methanol or water for all compounds that elicited a response, thus revealing that enhanced sensitivity and lower limits of detection are achieved with polar protic solvents. Response in acetone was equal to or slightly lower than response in acetonitrile in flow injection experiments; however, the water/acetonitrile and water/acetone mobile phases produced the better chromatographic separation. Water/methanol produced slightly less satisfactory separation but the greatest overall response. This increase in response was attributed to the protic nature of methanol and the elution of compounds in a higher organic mobile phase composition (retention times were ∼30% later in methanol). This work is intended to facilitate rational liquid chromatography/mass spectrometry method development for small molecule applications, including metabolomics.