This chapter describes two different methodologies used to improve the analytical performance of colorimetric paper-based biosensors. Microfluidic paper-based analytical devices (μPADs) have been produced by a stamping process and CO laser ablation and modified, respectively, through an oxidation step and incorporation of silica nanoparticles on the paper structure. Both methods are employed in order to overcome the largest problem associated with colorimetric detection, the heterogeneity of the color distribution in the detection zones. The modification steps are necessary to improve the interaction between the paper surface and the selected enzymes. The enhanced performance has ensured reliability for quantitative analysis of clinically relevant compounds.