The progress of N-alkylation reactions of poly(ethylene oxide) polyurethane conducting through a two-step reaction was monitored by In situ real-time infrared spectroscopy (In situ IR). Firstly, the starting polymer was metalaized with alkali metal catalyst and then the obtained urethane polyanion was treated with alkyl tosylate in DMSO/THF. In the case of 4,4-diphenylmethane diisocyanate (MDI)-based PEOPU, the absorption IR band of carbonyl at 1725 cm-1 (C=O) was shifted to 1628 cm -1 in the first step. which is due to the change of the electron environment from the formation of the anionic sites on urethane group. In second step, the band at 1628 cm-1 was shifted to 1702 cm-1, which is due to the disappearance of the anion and the formation of the N-alkyl urethane. A chemical conversion profile for post-polymerization N-alkylation reaction of PEOPU can be obtained from the monitoring of the change of the characteristic peak urethane carbonyl.