In this paper, a novel application of alternating penalty trilinear decomposition (APTLD) for high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been developed to simultaneously determine the contents of free amino acids in tea. Although the spectra of amino acid derivatives were similar and a large number of water-soluble compounds are coextracted, APTLD could predict the accurate concentrations together with reasonable resolution of chromatographic and spectral profiles for the amino acids of interest owing to its "second-order advantage". An additional advantage of the proposed method is lower cost than traditional methods. The results indicate that it is an attractive alternative strategy for the routine resolution and quantification of amino acids in the presence of unknown interferences or when complete separation is not easily achieved.