Drought is one of the key abiotic factors causing a loss in sugarcane output, which is one of the world's thirstiest crops. A mutant sugarcane callus line was used to create an in vitro selection system for drought resistance. The objective of this study was to obtain putative sugarcane mutant callus lines that had been chemically altered with sodium azide at various concentrations (0, 0.5, 1.0, and 1.5%). These lines were subsequently screened using high molecular weight PEG 6000 as a selection agent at various concentrations (0, 5, 10, 15, 20, 25, and 30%).  The experiment demonstrated a decline in the proportion of live callus and sensitivity index with increasing mutation concentration and PEG dose, with little to no live callus at the maximum mutation and PEG concentration. Biochemical investigation of the survived callus for proline accumulation and salicylic acid content revealed an elevated level of up to 20 percent PEG and 1.0 percent mutagen, followed by a quick drop as PEG and mutagen concentrations increased. The findings can be used to create drought-tolerant sugarcane lines and putative mutants, as well as to screen them in vitro.