Update publication information. In this work, we performed comprehensive glycosylation analysis to a candidate vaccine Spike protein. The Spike protein studied here was featured by a set of structural designs such as the six-proline substitution, furin cleavage site replacement, trimeric foldon incorporation as well as “hotspots” of SARS-CoV-2 variants. Besides, it was expressed in Chinese Hamster Ovary (CHO) Cell lines and manufactured at pilot scales, and thus, well represented latest variant-design vaccine of COVID-19. A combination of LC-MS/MS based analytical methods was dedicatedly applied to fully unravel the Spike N-glycosylation. Utilization of different protease was firstly explored and discussed. As a result, RPLC-MS/MS following Trypsin and GluC enzyme plus PNGase F treatment proved an effective and complementary method in analyzing N-glycosites and site-specific glycan. An HILIC-LC-MS based analytical method together with 2-AB labeling was also utilized to analyze N-glycan abundance for the Spike protein.