We investigated the use of separation, or substrate recovery, layers (SRLs), to enable the reuse of optical substrates after the deposition of multilayer reflective coatings, in particular Mo/Si multilayers as used for EUV lithography. An organic material (polyimide), known from other work to reduce the roughness of the substrate, was applied to the optical substrate. It appeared to be possible to remove the multilayer coating, including the SRL, without any damage or roughening of the substrate surface. The SRL was spin-coated at 1500 to 6000 rpm on different substrate types (Si, quartz, Zerodur) with diameters up to 100 mm. For this range of parameters, the multilayer centroid wavelength value remained unchanged, and its reflectivity loss on applying the SRL was limited typically to 0.7%. The latter was shown to be caused by a minor increase of the SRL surface roughness in the high-spatial-frequency domain. The roughness, characterized with an atomic force microscope, remained constant at 0.2 nm during all stages of the substrate recovery process, independent of the initial substrate roughness.