This work demonstrates the fabrication of microfluidic paper-based analytical devices (µPADs) suitable for the analysis of sub-microliter sample volumes. The wax-printing approach widely used for the patterning of paper substrates has been adapted to obtain high-resolution microfluidic structures patterned in filter paper. This has been achieved by replacing the hot plate heating method conventionally used to melt printed wax features into paper by simple hot lamination. This patterning technique, in combination with the consideration of device geometry and the influence of cellulose fiber direction in filter paper, led to a model µPAD design with four microfluidic channels that can be filled with as low as 0.5 µL of liquid. Finally, the application to a colorimetric model assay targeting total protein concentrations is shown. Calibration curves for human serum albumin (HSA) were recorded from sub-microliter samples (0.8 µL), with tolerance against ±0.1 µL variations in the applied liquid volume.