Previously, we suggested that mixed polyethyleneglycol (PEG)-modification of liposomes with mixture of 1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (PEG-DSG) with short and long polyoxyethylene chains led to increasing fixed aqueous layer thickness (FALT) and was useful in vivo. FALT is expected to be one of the important factors that influence the pharmacokinetics of liposomes. In this study, we investigated the connection between FALT and some parameters in vitro. In both mixed and single PEG-modified liposomes, the incorporation ratios of PEG-DSGs were the same, the residual amount of PEG-DSGs with serum proteins was not affected by molecular weight. Consequently, the effective properties in vivo of mixed PEG-modification may be the increase in the absolute amount of PEG-DSGs on the liposome membranes, decrease of slipping out action taken PEG-DSGs with long polyoxyethylene chains by serum protein, and thus maintenance of FALT. While, it has been suggested that mixed PEG-modified liposomes are effective in vitro, too, because of the lower leakage and same level of doxorubicin (DOX) uptake as plain liposomal DOX.