Yuan Li’s team demonstrated the formation of ice-like two-dimensional (2D) water flakes on self-assembled monolayers (SAMs) supporting cytochrome c (Cyt C) proteins under ambient conditions and without nanoconfinement. Using AFM, nano-AFM-IR, and IR/Raman spectroscopy, they directly visualized and chemically characterized the 2D flakes, showing controllable melting and growth behavior. The formation of these flakes was attributed to a twofold higher activation energy for water desorption from Cyt C compared to the SAMs. This study offers a new strategy to probe protein–water interactions and interfacial ice formation mechanisms in bio-relevant systems.