The use of plant growth regulators is one effective solution to improve sugarcane yields and sugar content in several countries. In this study, we examined the role of gibberellin acid (GA3) and glyphosate (Gly) plant growth regulators to determine the appropriate concentration of GA3 and Gly to increase the yield of sugarcane and sugar accumulation, respectively. The statistical results showed that GA3 was sprayed at 150 ppm to increment the actual yield by 19.94%; sucrose accumulation increased by 2.21%. With Gly treatment, although the yield decreased by 3.17%, sucrose accumulation increased by 11.27% compared to control trials. In this study, the combined concentration of 150 ppm of both GA3 and Gly gave the best results, for which sucrose accumulation increased from 2.21% to 10.74% and from 19.94% to 20.97% for actual yield. The results led to increased net income compared to the control. To address concerns about residues of plant growth regulators, residues of GA3 and Gly were evaluated after the sugarcane harvest using the HPLC and UV-vis methods, respectively. The analyzed results showed that their residues were lower than what is permitted in several countries. This showed the applicability of the study, on a large scale, to increase sucrose accumulation, productivity of sugarcane, and profit for farmers.