tThis 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 bethe 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.