In manufactured microfluidic channels, geometrical imperfections resulting from the manufacturing process are challenging in terms of reliability and functionality of the microfluidic device. In this work, a methodology for the geometrical and dimensional characterization of microfluidic channels using quantitative 3D optical microscopy is proposed. The methodology is based on a holistic approach including the identification of functionally relevant geometrical features and critical dimensions at design stage, as well as the related verification process at the manufacturing stage. At design stage, the critical geometrical characteristics are identified on the basis of their relation with the performance of the microfluidic device. Verification is based on a novel methodology in which measurement results collected by a surface topography measuring instrument are processed on the basis of concepts and procedures commonly applied to data generated by a coordinate measuring system. The method is presented using as an example the characterisation of a microfluidic channel realized by soft lithography and constituting part of a membrane micro valve. The geometry of the microfluidic channel is acquired by using an imaging confocal microscope.