The detailed understanding of the conformational pathway of fluticasone, a widely prescribed medicine for allergic rhinitis, asthma, and chronic obstructive pulmonary disease (COPD), from formulation to its protein-bound state, has been limited due to a lack of access to its high-resolution structures. The three-dimensional (3D) structure of fluticasone furoate 1 remains unpublished, and the deposited structure of fluticasone propionate 2 could be further refined due to refinement against new data. We applied microcrystal electron diffraction (MicroED) to determine the 3D structures of 1 and 2 in their solid states. The preferred geometries in solution were predicted by using density functional theory (DFT) calculations. A comparative analysis of the structures of 1 and 2 across three states (in solid state, in solution, and protein-bound conformation) revealed the course of the conformational changes during the entire transition. Potential energy plots were calculated for the most dynamic bonds, uncovering their rotational barriers. This study underscores the combined use of MicroED and DFT calculations to provide a comprehensive understanding of conformational and energy changes during drug administration. The quantitative comparison also highlights the subtle structural differences that may lead to significant changes in the pharmaceutical properties.