Red light sensing phytochromes typically have at least two functionally distinct conformations, a red-absorbing Pr state and a far-red-absorbing Pfr state. Understanding the structural differences between these functional states is key to appreciating aspects of light signal integration at the molecular level. Through potentially different interactions with downstream effectors, they can also lay the foundation for gaining better insights at an organismal level. The multidomain architecture of many full-length phytochromes makes them challenging targets for detailed structural analysis using classical X-ray crystallography or nuclear magnetic resonance approaches. While recent advances in cryo-electron microscopy offer a promising alternative, the inherently dynamic nature of photoreceptors can also be challenging for single-particle analysis. In this chapter, we describe an alternative approach for the structural analysis of phytochromes, hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS). This in-solution technique permits investigation of the conformational dynamics of proteins via the readout of their secondary structure stability. The ease of using different light regimes to enrich either Pr or Pfr states in solution is illustrated using the model bacteriophytochrome system from Deinococcus radiodurans. However, HDX-MS also offers the possibility of extracting the corresponding structural information from ensembles with partial Pr or Pfr states.