Molecular interactions and reactions in living cells occur with high concentrations of background molecules and ions. Many research studies have shown that intracellular molecules have characteristics different from those obtained using simple aqueous solutions. To better understand the behavior of biomolecules in intracellular environments, biophysical experiments were conducted under cell-mimicking conditions in a test tube. It has been shown that the molecular environments at the physiological level of macromolecular crowding, spatial confinement, water activity and dielectric constant, have significant effects on the interactions of DNA and RNA for hybridization, higher-order folding, and catalytic activity. The experimental approaches using in vitro model systems are useful to reveal the origin of the environmental effects and to bridge the gap between the behaviors of nucleic acids in vitro and in vivo. This paper highlights the model experiments used to evaluate the influences of intracellular environment on nucleic acid interactions.