This study describes a microfluidic paper-based analytical device (mu PAD) for separating plasma from whole blood and measuring glucose concentration. A two-dimensional mu PAD was fabricated by wax printing, using chromatographic paper and was functionalized by the incorporation of chitosan into the plasma separation zone. Red blood cells were isolated from whole blood in the plasma separation zone consisting of a chitosan polymer structure and a wax barrier. The separated plasma, with buffer, was injected into the test zone to cause a color change. The colorimetric results were digitized with a scanner, and the concentration was calculated using a calibration curve. This mu PAD, which separated the plasma from whole blood without a separation membrane, will be useful for constructing point-of-care testing devices that detect analytes in small sample volumes.