This study describes an extremely low-cost method for separating plasma in a sample of whole human blood on salt functionalized paper by means of osmotic pressure. When a sample of whole blood was introduced onto the salt functionalized paper, plasma dissolves the salt and places the red blood cells (RBCs) in a hypertonic medium. This leads to the generation of osmotic pressure across the cells membrane, and also the crenation of RBCs. The effect of different concentrations of salt on RBC deformation and crenation has been monitored using confocal microscopy. Depending upon the salt concentration, RBCs deform into various shapes under osmotic pressure. At high salt concentration, RBCs turn into deflated thin disks. This increases the RBCs contact with one another and with fibres in the paper as well. Besides, the counter ion valency charge of the Na+ suppresses the thickness of the charged double layer of RBC. Subsequently, aggregation of the deflated RBCs occurs. The aggregates are large enough to be separated chromatographically from the plasma phase of the wicking front. Our results show that 0.5 ?L addition of 0.68 M (4 w/v) saline solution (NaCl) can provide sufficient plasma separation on a filter paper for diagnostic applications. A colorimetric blood glucose concentration assay is employed to demonstrate the efficiency of this plasma separation method on paper. The experimental investigation indicates that although the crenation of RBCs forced a small amount of water into plasma, this method is suitable for performing glucose assay in human blood on paper. Our method can enhance bioassays performed on microfluidic paper-based analytical devices (muPADs) by combining the separation and testing of plasma into a single device with no significant additional cost.