Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under favorable condition. Although many endogenous RNAs contain sequences that can readily fold into RNA G4s (rG4s) in vitro, the regulation of their folding and functions in vivo, is not well understood. Here, we show that the folding of putative rG4s in human cells into bona fide rG4 structures is dynamically regulated by stress. By using a high-throughput approach based on differential reactivity of dimethyl sulfate (DMS) towards Gs within folded vs unfolded rG4s, we identified hundreds of endogenous rG4s whose folding is promoted by cellular stress and validated them by using a newly developed rG4-specific ligand. Stress-dependent rG4s are enriched in mRNA 3′ untranslated regions (3′-UTRs), suggesting their role in regulating mRNA stability under stress. Lastly, rG4 folding is reversible upon stress removal. In summary, our study provides insights into dynamic regulation of rG4 folding in living human cells and suggests that rG4s may function as prevalent regulatory elements with potential roles in regulating mRNA stability and cellular stress response. Finally, we also demonstrated that specific RNA G-quadruplexes derived from transfer RNAs are assembled under stress. Thes tRNA-derived rG4s are pro-survival and trigger cytoprotective response. We propose to use synthetic analogs of these molecules as therapeutic agents in human disease such as neurodegenerative conditions, e.g., Amyotrophic Lateral Sclerosis.