Display omitted ► Solid-state NMR provides a sensitive tool for analyzing steroid hormone conformation. ► DHEA and spironolactone reveal multiplet pattern in 13C NMR spectra. ► DHEA and spironolactone undergo a conformational change in lipids. ► DHEA and spironolactone are flexible and can adopt slightly different conformations. Solid-state 1H/ 13C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to two steroid compounds: dehydroepiandrosterone (DHEA) and spironolactone (SPI), to analyze their conformations at the atomic level. In the absence of lipid, the high-resolution 13C CP/MAS NMR signals of DHEA and SPI in a powder form reveal multiple patterns, with splittings of 30–160 Hz, indicating the existence of multiple conformations. In the mimic lipid environment formed by mixing 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl- sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, the resulting DHEA and SPI spectra revealed mostly singlet patterns, suggesting that these steroids undergo a conformational change leading to a specific conformation in the lipid environment. Evidence from chemical shift isotropy and anisotropy analysis indicates that DHEA might adopt conformations subtly different from that seen in solution and in the powder form. In conclusion, we demonstrate by solid-state NMR that the structures of DHEA and SPI may adopt slightly different conformations in different chemical environments.