Proteins are practically never in a buffer-free solution. We therefore studied the effects of some important biological buffers 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) on the lower critical solution temperature (LCST) phase transition of poly(N-isopropylacrylamide) (PNIPAM), an isomer of polyleucine, as a model compound for protein. The results from dynamic light scattering (DLS) analysis showed that the LCST of PNIPAM aqueous solutions were decreased significantly with increasing the buffer concentration, due to the presence of the buffers destroying the hydration structures and subsequent aggregation of PNIPAM. Based on density functional theory (DFT) and molecular mechanics calculations, these buffers are highly polar compounds and, therefore, strongly interact with water molecules, causing buffering out of PNIPAM. The buffer's affinity to water follows the order MOPS > MES > MOPSO. The LCST values determined from DLS follow the order MOPSO > MOPS approximate to MES. The Fourier transform infrared spectra (FTIR) reveal that no direct binding between the buffer and the polymer occurs but interacts with the first hydration shell of the polymer. In comparing with the MOPS buffer, the unexpected effects on the PNIPAM were found in the MOPSO-containing system due to MOPSO has one more OH group. This group interacts with water clusters around the hydrophobic isopropyl groups, which changes the hydration state of the polymer. It also should be noted that the phase transition in the presence of buffers is similar to those observed with kosmotropic salts and sugars (protein stabilizers) in terms of lowering the LCST and the dehydration of PNIPAM groups. Such favoring of the compact globule state observed at high buffer concentrations will provide a protective effect against denaturation of globular proteins. The interactions of the studied buffers with bovine serum albumin (BSA) have been investigated and found that these buffers stabilized BSA, based on DLS, FTIR, and UV-vis absorption studies. The present study will act as a key for the lock of buffer and macromolecule mechanism.