The collagen layer of the human tympanic membrane (TM), which is essential for sound conduction and hearing, can be assessed in vivo by using endoscopic polarization-sensitive optical coherence tomography (PS-OCT), as we have shown recently. Since the resolution of the utilized system was limited, an ex vivo validation of the structural information is presented here. By comparing higher resolution PS-OCT images of a temporal bone specimen and polarized light microscopy (PLM) of histological slices from that TM with picrosirius red staining, the microstructure is investigated.