Glyphosate, the active ingredient in many herbicides, has been widely used in agriculture since the 1970s. Despite initial beliefs in its safety for humans and animals due to the absence of the shikimate pathway, recent studies have raised concerns about its potential health effects. This study aimed to identify glycomic changes indicative of glyphosate-induced toxicity. Specifically, the study focused on profiling -glycosylation, a protein post-translational modification increasingly recognized for its involvement in various disorders, including neurological conditions. A comprehensive analysis of rat serum -glycomics following chronic exposure to glyphosate-based herbicides (GBH) was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results revealed significant changes in the -glycan profile, particularly in sialylated and sialofucosylated -glycans. The analysis of -glycans across gender subgroups provided insights into gender-specific responses to GBH exposure, with the male rats exhibiting a higher susceptibility to these -glycan changes compared to females. The validation of significantly altered -glycans using parallel reaction monitoring (PRM) confirmed their expression patterns. This study provides novel insights into the impact of chronic GBH exposure on serum -glycan composition, with implications for assessing glyphosate toxicity and its potential neurological implications.