Adsorbed and immobilized proteins play an important role in a variety of biological phenomena of industrial importance. Protein adsorption onto solid surfaces significantly alters subsequent biological responses to the surface. Proteins at solid-liquid interfaces can therefore be exploited to design surfaces that influence or direct the response of the biological environment. Several analytical tools have been applied to examine the state of the adsorbed proteins, that is, their identity, concentration, conformation, orientation, and spatial distribution. These characteristics play important roles in the biological response to solid surfaces. This article reviews the application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) for characterizing the state of adsorbed protein films. Due to the analytical power of this technique, ToF-SIMS provides wealth of information on the state of the adsorbed proteins. The capabilities and limitations of ToF-SIMS for examining the state of adsorbed proteins will be discussed along with future directions for using this technique to characterize adsorbed and immobilized proteins. (C) 2004 Elsevier B.V. All rights reserved.