The effects of trimethylamine N‐oxide (TMAO) on guanidinium chloride and hydrogen‐bonded networks of water are explored in this joint Raman spectroscopic and quantum chemical study. Both TMAO and guanidinium are osmolytes that affect the stability of proteins, as TMAO is known to stabilize and counteract the destabilizing effects of guanidinium. While guanidinium is very similar in chemical structure to urea, the exact mechanisms of the molecular interactions between guanidinium, TMAO, and proteins continue to be investigated. Herein, we use Raman spectroscopy to elucidate the physical interactions between TMAO and guanidinium in aqueous solutions to better understand how these important osmolytes interact with each other and affect adjacent hydrogen‐bonding networks of water. Comparing experiment to theory yields good agreement and allows for the identification and tracking of different vibrational modes. It was determined that adding TMAO into an aqueous solution of guanidinium induces a blue shift (shift to higher energy) in guanidinium's H‐N‐H bending modes, which is indicative of direct interactions between the two osmolytes and similar to the earlier results observed for TMAO interacting with urea.