Hydrophobic matching between transmembrane protein segments and the lipid bilayer in which they are embedded is a significant factor in the behavior and orientation of such transmembrane segments. The condition of hydrophobic mismatch occurs when the hydrophobic thickness of a lipid bilayer is significantly different than the length of the membrane spanning segment of a protein, resulting in a mismatch. This mismatch can result in altered function of proteins as well as nonnative structural arrangements including effects on transmembrane α-helix tilt angles, oligomerization state, and/or the formation of non-transmembrane topographies. Here, a fl uorescence-based protocol is described for testing model transmembrane α-helices and their sensitivity to hydrophobic mismatch by measuring the propensity of these helices to form nontransmembrane structures. Overall, good hydrophobic matching between the bilayer and transmembrane segments is an important factor that must be considered when designing membrane proteins or peptides.