Testosterone is a representative natural steroid hormone with potent androgenic and anabolic properties and consequently used by youngsters and athletes to achieve bodies with lean muscle mass, improve skin elasticity as well as increase endurance and tolerance to strenuous exercise. The long-term use of marketed preparations containing testosterone can result in latent health risks to consumers. There are genuine demands for continuous development of highly sensitive and effective quantification methods for analytically monitoring the illegal use of testosterone. Previously existing methods like HPLC-UV, HPLC-MS and GC-MS are time consuming as well as tedious operation procedures limit their use. Hence a simple, precise, accurate, sensitive and reliable method for determination of low concentrations of testosterone in biological fluids viz., serum has been developed. The developed spectrofluorimetric method provides a faster and low cost quality control by simultaneous routine analysis of testosterone in biological fluids as well as marketed preparations containing testosterone.