In conjunction with surface chemistry, the mechanical properties of cell culture substrates provide important biological cues that affect cell behavior including growth, differentiation, spreading, and migration. The phenomenon has led to the increased use of biological and synthetic polymer-based flexible substrates in cell culture studies. However, widely used methods for measuring the Young's modulus have proven difficult in the characterization of these materials, as they tend to be relatively thin, soft, hydrated, and tethered to glass substrates. Here we describe three methods that have been applied successfully to probe the flexibility of soft culture substrates.