The admission into and diffusion through nanoscale pores by molecules is a fundamental process of great importance to biology and separations science. Systems (e.g., chromatography, electrophoresis) designed to harness such processes tend to remove the separation process from the detection event, both spatially and temporally. Here, we describe the preparation and characterization of thin optical Fabry-Pérot films of mesoporous silica (pSiO(2)) that can detect protein infiltration by optical interferometry, which probes the separation process in real time and in the same ultrasmall physical volume (5 nL). Admission of a protein into the pores is controlled by the diameter (∼50 nm) and the surface charge of the pores, and both the rate and the degree of protein infiltration are a function of solution pH. Test proteins bovine serum albumin (BSA, 66 kDa), bovine hemoglobin (BHb, 65 kDa), and equine myoglobin (EMb, 18 kDa) are admitted to or excluded from the nanophase pores of this material based on their size and charge. The rate of protein transport within the pores of the pSiO(2) film is slowed by 3 orders of magnitude relative to the free-solution diffusion values, and it is maximized when pH = pI.