Neither the adsorbed amount per unit actual surface area nor the structural stability of hen egg lysozyme is influenced by increasing the nanometer-scale roughness (5 < R-ave < 60 nm) of a series of model substrates. Seven poly(ether sulfone) (PES) ultrafiltration membranes of increasing mean pore size with the same surface chemistry were chosen as model rough surface substrates. Topographical images, using atomic force microscopy, combined with attenuated total reflection Fourier transform infrared spectroscopy (ATR/ FTIR) and sessile captive bubble contact angle measurements were used to characterize the surface properties of the substrates. ATR/FTIR spectroscopy together with a newly developed optimization algorithm for predicting the content of secondary structure motifs is used to correlate the secondary structure and amount of adsorbed lysozyme with the substrate surface roughness. From the adsorption measurements, the net adsorbed amount (total minus nonspecific adsorbed amount) of lysozyme corresponded to approximately one monolayer of coverage for all the substrates independent of the roughness. Although lysozyme was structurally disturbed through adsorption to PES substrates, no significant changes in its secondary structure were observed with the increasing roughness.