Growth and mosquitocidal activity of Lysinibacillus sphaericus 9B24 was studied in defined medium supplemented with amino acids. It was found that L-glutamate, L-arginine, and L-proline enhanced the sporulation and mosquitocidal activity of the tested culture against Culex pipiens larvae. Different concentrations of these three amino acids were tested for sporulation and toxin formation by Lysinibacillus sphaericus 9B24 in comparisonwith Lysinibacillus sphaericus 2362.Optimumconcentrations were 0.5-0.75%for L-arginine and 0.75%for L-glutamate and L-proline.All tested mixtures of these amino acids enhanced the sporulation and mosquitocidal toxicity of both cultures. L-arginine- L-proline - L-glutamate mixture at their optimum concentrations was the most mosquitocidal enhancer for Lysinibacillus sphaericus 2362 (LC50 1.5 × 10-5) however, mixture of L-proline- L-glutamate was the optimum for Lysinibacillus sphaericus 9B24 (LC50 1.66 × 10-5). Itwas concluded that thewasteswhich contain high quantities of L-arginine, L-proline and L-glutamate could be used as media for cost effective production of Lysinibacillus sphaericus.