Abstract: As the secondary metabolites of plants, flavonoids are very important active ingredients in natural medicinal plants and have been proved to possess the extensive biological activities. It has been reported that flavonoids can bind to plasma proteins. Lysozyme is a globular protein and can interact with many small molecules for therapeutic applications. Therefore, the studies of interactions between flavonoids and proteins are not only helpful for understanding of the biological action of small natural organic molecules, but also are beneficial for the development of novel drug candidates. Native electrospray ionization mass spectrometry has been widely applied in the studies of the interactions of proteins and small molecules. During the electrospray ionization (ESI) analysis, some organic solvents are often used as a cosolvent. However, it is not clear for the effects of addition of some organic solvents on the ESI analysis of protein-ligand complexes. The solvent dimethyl sulfoxide (DMSO) is one of cosolvents. There are few studies on the effect of DMSO on protein-small molecule interactions by electrospray mass spectrometry (ESI-MS). Here, the effects of DMSO on the ESI-MS analysis of four lysozyme-flavonoids of icariin, rutin, naringin and scutellarin complexes were investigated. The stable, labile and non-specific binding protein complexes with small molecule ligands were determined by ESI-MS, respectively. It was found that the content of DMSO affects the apparent binding constants of lysozyme and small molecule ligand complexes. The low amounts of DMSO lead to increase the apparent affinity of the labile lysozyme-flavonoid complexes to some extent. It also can stabilize the lysozyme complex with N,N',N''-triacetylchitotriose even under the high capillary temperature. The addition of DMSO cannot lead to the non-specific binding of lysozyme complex with maltose. In addition, the content of DMSO also affects the charge states of the lysozyme complexes. Compared with the sample without DMSO, the charge states of the complex initially decrease with the addition of DMSO. Then the charge states of the complex increase with the further increasing content of DMSO and even increase to higher charge states than those without DMSO. Therefore, it is indicated that the addition of DMSO can affect the ESI-MS analysis of the interaction of protein and small ligands, including the apparent binding constants and charge states of protein-ligand complexes. For the labile lysozyme-flavonoid complexes in ESI-MS, DMSO at the optimized content is shown to stabilize these complexes during ESI-MS analysis. It was suggested that the amount of DMSO used in ESI-MS should be carefully controlled.