Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile-phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.