Recently an algorithm to separate fluorescent and reflective components from a hyperspectral image has been reported, in which the important task of spectral unmixing of multiple fluorescent components was left unresolved. In this paper, we present the algorithm to simultaneously separate those components and unmix fluorophores (SSUF: Simultaneous Separation and Unmixing of Fluorescent components) from a single hyperspectral image. Two variants are introduced for the cases when fluorophore spectra are known and unknown. Experimental results confirm the validity of the proposed method.