Chemoresistance largely hampers the clinical use of chemo drugs for cancer patients, combination or sequential drug treatment regimens have been designed to minimize chemotoxic and resensitize chemoresistance. In this work, the cytotoxic effect of cisplatin was found to be enhanced by palbociclib pretreatment. With the integration of proteomic and N-glycoproteomic workflow, we found that palbociclib alone mainly enhances the N-glycosylation alterations in Hela cells, while cisplatin majorly increases the DEPs (different expression proteins) including apoptosis proteins. As a result, the sequential use of two drugs induced a higher expression level of apoptosis proteins BAX and BAK. Those altered n-glycoproteins induced by palbociclib were implicated in pathways that are closely associated with cell membrane modification and drug sensitivity. Specifically, the top 4 frequently glycosylated proteins FOLR1, L1CAM, CD53, and LAMP1 were all drug resistance or drug sensitivity associated proteins located mainly in the cell membrane. It is suspected that palbociclib-induced N-glycosylation on the membrane protein allowed the Hela cell to become more vulnerable to cisplatin. Our study provided new insights into the mechanism underlying the sequential use of target drug and chemotherapy drug, meanwhile suggesting a high-efficiency approach that involves proteomic and N-glycoproteomic to facilitate drug understanding.