DMSO is the standard solvent for preparing stock solutions of compounds for drug discovery. The assay concentration of DMSO is normally 0.1% to 5% (v/v) or 14 to 715 mM. Thus, DMSO is often one of the principal additives in assay buffers. This standardization of stock solutions does not eliminate possible pitfalls associated with the effects of the DMSO-containing solutions on individual proteins. In this article, the authors want to emphasize the importance of detailed studies of these effects in the early stages of drug discovery. Two protein systems, the extracellular soluble domain of the human growth hormone receptor (hGHbp) and the phosphatase domain of PFKFB1 (BPase), were used for the study on effects of DMSO on protein stability, protein aggregation, and binding of drug compounds. The study revealed significant differences in the proteins' behavior in the presence and absence of low amounts of DMSO. The addition of DMSO resulted in destabilization of the proteins investigated and also changed the apparent binding property of 1 protein. The authors have also shown that low DMSO concentrations influence the ionization process in electrospray ionization mass spectrometry (ESI-MS).