Laser-induced culture fluorescence is one of the few nondestructive measurements that are currently available for the on-line monitoring of the internal state of a microorganism. Many biomolecules, including NADH, exhibit characteristic fluorescence spectra that are dependent on the excitation wavelength. Many fluorophores are simultaneously present in a typical fermentor, and the fluorescence spectra for a mixture of fluorophores must be deconvoluted to yield individual concentrations, after the noisy spectral data are treated in the Fourier domain. Because of the secondary optical effects such as the repeated re-absorption and re-emission of light in a mixture, the principle of superposition does not hold true. Furthermore, the overall emitted signals are shifted toward the higher wavelengths. A rigorous nonlinear model is developed for the most commonly used optical arrangement to relate the detected signal level to the fluorophore concentration. As a model system, the emission spectra of binary and ternary mixtures of simple aromatic amino acids (tyrosine, tryptophan, and phenylalanine) are analyzed with a range of computer algorithms. The predictions resulting from different analytical approaches are compared to the true component concentrations, and the effectiveness of each method is evaluated. In general, it is most difficult to detect a small amount of one component either in the presence of larger concentrations of other fluorophores or in the presence of an unknown fluorophore. The presence of an unknown fluorophore is determined by spectral matching and hypothesis testing. The application of culture fluorescence measurements to fermentation control will be discussed.