In this study, we delved into deciphering the glycosylation patterns and precise localization of glycosylation sites within CAR-T proteins. Specifically, we focused on samples derived from human T cells that underwent gene knockout of AAVS1 and SPPL3, two critical genes that could potentially influence CAR-T function. To ensure accuracy and precision, we employed optimal tribrid orbitrap mass spectrometry settings for glycosylation landscapes of the CAR-T proteins. By analyzing these data, we aimed to gain a deeper understanding of how AAVS1 and SPPL3 knockout impacted the glycosylation profiles and ultimately the function and efficacy of CAR-T cells in immunotherapy applications.