Environmental contamination by antibiotics not only perturbs the ecological balance but also poses a risk to human health by promoting the development of multiantibiotic-resistant bacteria. This study focuses on identifying the biochemical pathways associated with tetracycline (TC) transformation/degradation in vetiver grass that has the potential to be used as a biological remediation system in TC-contaminated water sources. A hydroponic experimental setup was used with four initial TC concentrations (0, 5, 35, 75 ppm), and TC uptake was monitored over a 30-day period. Results show that TC transformation in the media occurred during the first 5 days, where a decrease in the parent compound and an increase in the concentration of the isomers such as epitetracycline (ETC) and anhyrotetracycline (ATC) occurred, and TC disappeared in 20 days in tanks with vetiver grass. However, the isomers ETC and ATC remained in the control tanks for the duration of the trial. Transformation products of TC in plant tissue were analyzed by using ultra HPLC high-resolution Orbitrap mass spectrometery (HRMS/MS), which indicates amide hydrolysis of TC in vetiver roots. Metabolic profiling revealed that glyoxylate metabolism, TCA cycle, biosynthesis of secondary metabolites, tryptophan metabolism, and inositol phosphate metabolism were impacted in vetiver root by TC treatment.