This article describes the development of a simple, portable assay system using microfluidic paper-basedanalytical devices (PADs) coupled with colorimetric detection for rapid measurements. The proper-ties of different paper substrates were first investigated to determine which type of paper would be the most suitable for the fabrication of the PADs. Simultaneous detection of horseradish peroxidase(HRP) utilizing a 5 L sample analytical volume was demonstrated using a single PAD. Hydrophilictest regions were separated by hydrophobic barriers, which were fabricated through photolithography.These test regions were immobilized with 10 mM of 3,3,5,5-tetramethylbenzidine for HRP assay. Thedetection range obtained with the proposed system covered HRP concentrations from 0.37 to 124 fmol (or3–1000 ng mL−1). The detection limit (blank + 3) for HRP was calculated to be 0.69 fmol (or 5.58 ng mL−1)through a 4-parameter logistic nonlinear regression using results obtained within a 15 min assay time.The findings obtained using the developed system suggest that PAD assay systems for simple buthighly sensitive measurements can be designed to give on-site determinations of target compoundsusing peroxidase-conjugated molecules.