One of the essential points in analytical chemistry and in natural sciences is the presence of a linear calibration line; i.e., only is the linear calibration line able to determine the chemical and biochemical molecules. High-recovery condition is required to obtain the linear calibration line in high-performance liquid chromatographic (HPLC) protein determination. In this regard, however, reversed-phase HPLC (RP-HPLC) determination of proteins seems to be limited to some non-glycosylated proteins. On the other hand, high-performance gel-permeation chromatography (HPGPC) seems more conveniently applicable to proteins and glycoproteins. In this review reliable HPGPC protein determination method is described. The method is applicable to wide variety of biological specimens, such as human body- fluids, rodent plasma, tissue homogenates, and cultured cells in high sensitivity.