Rationale Ammonium fluoride has been shown to improve sensitivity in electrospray ionization in mass spectral detection when used as a mobile phase additive. Internal investigation showed sensitivity improvement for steroid compounds with LCMS. Our work presented here investigates ammonium fluoride as a mobile phase additive in improving mass spectral ionization compared with other mobile phases under fully optimized instrument conditions. Methods For our research, we investigated ammonium fluoride in comparison with formic acid and ammonium hydroxide as mobile phase additives to measure sensitivity effects in electrospray ionization sensitivity. Full source optimization was performed for nine commercially available compounds at three different organic concentrations (30, 60, or 90%) with formic acid, ammonium fluoride, and ammonium hydroxide adjusted mobile phase sets. Optimization results were compiled to generate individual methods by compound, polarity, mobile phase additive, and organic concentration, respectively. These methods were used for final flow injection analysis to objectively compare compounds analyzed with the different solvent systems under optimal conditions for each mobile phase composition. Results Negative ESI data showed 2 – 22-fold sensitivity improvements for all seven compounds with ammonium fluoride. Positive ESI data showed > 1 - 11-fold improvement in sensitivity for four of seven compounds and equivalent (or slight drop in) sensitivity for three of seven compounds with ammonium fluoride adjustment. Acetaminophen showed the greatest sensitivity improvement in both polarities, with 7-22-fold (ESI-) and 4-11-fold (ESI+) improvements in sensitivity, respectively Conclusions With fully optimized source conditions there is good likelihood of improving MS sensitivity (ESI-) in using ammonium fluoride adjusted mobile phase. Investigation with ESI+ analyses showed mixed results, with four of seven compounds showing improvement and others showing equivalency or slight loss in sensitivity. Our investigation focused solely on sensitivity gains achievable in ESI-MS by source optimization and mobile phase additive selection by flow injection without chromatography.