The creatine. creatinine cyclization, an important metabolic phenomenon has been initiated in vitro at acidic pH and studied through Raman spectroscopic and DFT approach. The equilibrium compositionof neutral, zwitterionic and protonated microspecies of creatine has been monitored with time as the reaction proceeds. Time series Raman spectra show clear signature of creatinine formation at pH 3 after similar to 240 min at room temperature and reaction is faster at higher temperature. The spectra at pH 1 and pH 5 do not show such signature up to 270 min implying faster reaction rate at pH 3. (C) 2014 Published by Elsevier B.V.