Chrysopogon zizanioides (L.) Nash is widely used in Ayruveda for treatment of various ailments. Despite numerous in vitro protocols, no report had studied the chemical composition of the in vitro shoots culture. Hereto, shoots were induced from crown explants on MS (Murashige & Skoog) medium and propagated on MS (0.5 mg IAA/L) medium. Various additives levels of phenylalanine (Phe) and proline (Pro), alone or in different combinations with salicylic acid (SA), polyethylene glycol (PEG) were tested. Highest shoot multiplication was attained on MS (100 mg/l Phe + 500 mg/l PEG). Highest yield of total flavonoids (32.82 ± 0.08 catechin equivalent) and phenolics (286.76 ± 1.55 gallic acid equivalent) were achieved on MS (100 mg/l Phe + 100 mg/l SA) and MS (50 mg Pro/l), respectively. Shoots from these two media were chemically profiled in comparison to the control media using LC-MS/MS. Nine flavone glycosides, three lignans and flavolignans, four phenolics, three carboxylic acids and three fatty acids were the most abundant in the three extracts. Treatments and elicitors didn't affect the qualitative composition of shoot extracts but did influence the polyphenolics yield. This study paves the way for more research on in vitro biosynthesis and production of bioactive phytochemicals in vetiver shoots.