By means of two alternative methods lipid and protein hydroperoxides (HP) were determined by fluorometry using the diphenyl-1-pyrenylphosphine (DPPP) probe. It has been shown that the formation of the fluorescence was influenced by the type of solvent and HP whereas the presence in the media of antioxidants such tocopherol and butylated hydroxytoluene had no effect. The combination of the chloroform:methanol (2:1, v/v) solvent mixture that is widely used for lipid extraction was combined with suitable solvents to develop a method with the maximum performance in determining HP in lipid extracts. Using a variety of lipids and lipid extracts, the final method proposed agreed well with the thiocyanate method for HP determination. In addition, the DPPP method was very sensitive, precise, accurate, free of interferences and specific for the determination of lipid soluble HP. DPPP can be also used to measure HP soluble in hydroalcoholic media. This alternative procedure showed a similar performance to its lipid soluble equivalent and was able to measure hydrogen peroxide promoted peroxidation of bovine serum albumin and water soluble HP in protein extracts. With the addition of triphenylphosphine the hydroalcoholic method is specific for the determination of protein HP.