Stripe rust is a fungal disease that has devastated the barley production for a long time. The present study focused on the role of β-glucan, PR proteins, diamine oxidase (DAO), polyamine oxidase (PAO), key enzymes and metabolites of phenol and proline metabolism in the stripe rust resistance of barley. RD2901 with resistant behavior against stripe rust showed increased levels of PR proteins, phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL) along with the accumulation of β-glucan and lignin which strengthen the plant cell wall during plant-pathogen interaction. It also depicted the enhanced activities of glutamate dehydrogenase (GDH) and ornithine aminotransferase (OAT) coupled with the increased amounts of proline, glycine betaine and choline after infection with M-race of P. striiformis f. sp. hordei. On the contrary, the sensitive genotype Jyoti was unable to enhance the activities of most of these enzymes except PAL and OAT so that it showed an increase in lignin and choline contents only. Secondly, the increase in lignin content was less as compared to the tolerant genotype. Hence, it can be inferred that these key metabolites and enzymes of various metabolic pathways may contribute to the resistance of barley against stripe rust pathogen. This study suggested that these key enzymes and their metabolites could serve as markers for the characterization of plant defensive state that is essential for crop protection.