A novel microfluidic paper-based analytical device (mu PAD) utilizing a nitrocellulose (NC) membrane to detect IgG antibodies through a colorimetric analysis is described. The mu PAD was constructed using layered polyethylene terephthalate (PET) and pressure-sensitive adhesives (PSA). The biotin labeled Goat Anti-Mouse IgG antibody was spotted and dried on the NC channel prior to subjecting it to a series of wash solutions (Tris-tween), increasing concentrations of alkaline phosphatase conjugated to streptavidin (Strep-ALP), and paranitrophenyl phosphate (p-NPP) realizing a vibrant yellow color. The reaction proceeds for 10 min before applying the p-NPP stop solution. The device was then dried, scanned, and analyzed yielding a linear range of inverse yellow color intensities versus Strep-ALP concentrations. The development of this simple mu PAD should further facilitate the use of NC in colorimetric assays to detect and quantitate antibodies.