Leaf rust of wheat is an important biotic plant stress influence in yield reduction of wheat. Host resistance is a feasible approach for dealing with disease management in wheat. Germplasm screening with resistance reactions will be helpful in the development of resistant lines; on the other hand, plant cellular responses during the host pathogen interaction may vary depending on the resistance and susceptibility of the cultivar. Knowledge of cell membrane stability and stress protein during the pathogen interaction helps in quantifying disease and the effect of these parameters on yield losses. 48 germplasm has been screened with two susceptible germplasm, and two biochemical parameters such as proline and membrane stability were calculated among germplasm. Agra local and PBW343 show the lowest proline content at 0.505 and 0.405 μMoles/g, with the lowest membrane stability of 12.34 and 115.32 with susceptible reactions. Cluster analysis of the germplasm based on quantitative disease scoring and qualitative disease parameters divides the germplasm into four clusters, such as germplasm viz., cluster 1 with 16 germplasm viz., JSW 3, 5, 9, 8, 10, 11, 15, 16, 18, 19, 26, 28, 29, 32, 34, 41. This germplasm was observed with disease severity from 0.75 to 6, and the disease reaction of the germplasm ranges from R, RMR, and MR and was recorded as 1.85 to 2.475 μMoles/g, and EC or membrane stability was between 10.32 and 42. Observing the experimental studies, it was identified that the categorization of this germplasm into slow rust and race-specific based on the disease scoring and physio-biochemical parameters helps in the breeding of durable varieties.