This is the second in a series of papers in which we present methods demonstrated in our group for the estimation of transport properties in gas diffusion layers (GDLs) for proton exchange membrane fuel cells (PEMFCs). Here we describe a method for determining separately the in-plane (x, y-directions) and through-plane (z-direction), viscous and inertial permeability coefficients of macro-porous substrates and micro-porous layers by controlling the direction of the gas flow through the porous sample. The method is applied initially to the macro-porous substrate of the GDL alone and subsequently to the macro-porous substrate with different micro-porous layers applied on it. The permeability coefficients of the micro-porous layer are calculated from the two measurements. The permeability coefficients are calculated from the Darcy–Forchheimer equation by application of the method of least squares. The method was applied to GDLs having different contents of polytetrafluoroethylene (PTFE) and carbon types. The samples with a higher PTFE content have in-plane and through-plane viscous permeability coefficients higher than those of the samples with lower PTFE content. The in-plane and through-plane viscous permeability coefficients also depend on the carbon type.