An octadecylsilyl silica (ODS) column coated with phosphatidylcholine (PC) was prepared by using a dynamic coating method, and its separation characteristics were evaluated for direct sample injection analysis of biological fluids. First, fundamental separation behaviors of various kinds of large and small molecules/ions were examined by HPLC using the PC-coated ODS column with UV absorption detection, for which pure water was used as a mobile phase. In the experiments, proteins (large molecules) were rapidly eluted in the range of 2.07 min (dead time)-3.20 min, while small molecules, such as inorganic anions, amino acids, organic acids, and organic drugs, were eluted after 3.50 min. The fact that proteins and small molecules/ions were separated on the PC-coated ODS column suggested that the present column can be used for direct sample injection analysis of biological fluid samples, such as urine and blood serum. Thus, the PC-coated ODS column was used in the analysis of arsenic species in urine and in the analysis of nicotinamide, theophylline, tryptophan, and caffeine in a model blood serum solution without any sample pretreatment.