Total internal reflection fluorescence (TIRF) microscopy produces a thin excitation field (the evanescent field) that nominally decays exponentially. This field is ideal for selective excitation of fluorophores near the coverslip/sample interface. We present an experimental method, where the depth and axial profile of the evanescent field can be measured directly by microscopic observation of low refractive index fluorescently labeled spherical beads in an indexmatched solution. To demonstrate the technique, through-theobjective TIRF is set up with laser excitation. In this configuration, the axial profile of the evanescent field created by either a 1.45-numerical aperture (NA) or a 1.65-NA objective fits well to a double exponential. At the coverslip/sample interface, about 90% of the evanescent field is represented by an exponential with a decay rate consistent with that expected for a theoretical evanescent field; the remaining 10% of the field is represented by an exponential with a much longer decay constant and is identified as scattering. The approach presented here is particularly useful for investigating the quality and axial profile of the evanescent field in both laser-based and mercury arc-based through-the-objective TIRF systems where a significant amount of light scattering can occur in the illumination optics. © 2006 Society of Photo-Optical Instrumentation Engineers.