Abstract: Soybean phospholipids have many bioactivities.However, the poor solubility of soybean phospholipids in water limits its cellular biological efficacy.In this work, glycerol, dimethylsulphoxide (DMSO) and ethanol were used as the solvents to dissolve phospholipids and to explore the preparation method of soybean phospholipids in cell culture solution.Firstly, the optimal linear absorption wavelength was obtained by the whole wavelength scan and gradually reducing the wavelength range with microplate reader.Secondly, the solubility of soybean phospholipids in three solvents were investigated according to the standard curve of soybean phospholipids in hexane solution, and the particle size of soybean phospholipid-glycerol-aqueous solution was measured by Malvern Particle.Finally, the effect of soybean phospholipid-glycerol solution on the Caco-2 cells was detected by MTT assay.The results showed that at 340 nm, the absorbance of soybean phospholipid had the best linear relationship with its concentration.The solubilities of soybean phospholipid in ethanol, DMSO and glycerol were 28.8 mg/mL, 31.6 mg/mL and 180 mg/mL, respectively.The effect of soybean phospholipid-propanol solution on the Caco-2 cells was enhanced with the increase of its concentration.Therefore, glycerol was used as the soluble agent for soybean phospholipids in aqueous solution, which wald be used for the determination of soybean phospholipids in cell culture.