Poly-pyridobisimiazole (M5) single filaments subjected to varying degrees of heat treatment have been analyzed using Fourier Transform Infrared (FTIR) microspectroscopy in transmission mode to detect changes in the state of intermolecular hydrogen bonding as a function of fiber annealing conditions. The FTIR absorbance bands associated with hydrogen bonding in M5 fiber have been identified, and the integrated molar absorption coefficients for the bands of interest have been determined experimentally, which allows to quantify the concentration of N―H vibration groups hydrogen-bonded (H-bonded) to water molecules, and the concentration of N―H vibration groups H-bonded to adjacent polymer chains in the fiber. A dual mechanism kinetic rate expression is used to describe intermolecular H-bond formation in M5 fiber as a function of annealing conditions, from which an activation energy for H-bond formation of 14.8 kJ/mol is obtained.