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AbstractTwo-dimensional fluorescence spectroscopy (2D-FS) has been used as a new method for determining the viability of tobacco cells (Nicotiana tabacum L.). Both horizontal beam geometry and a vertical set-up achieved with bifurcated fibres were tested. The latter arrangement enabled us to avoid the negative effect of cell sedimentation. Incubation of a tobacco BY-2 cell suspension with dimethylsulfoxide (DMSO) (010% v/v) resulted in cell samples differing in their viability from fully viable (02% DMSO) to totally non-viable (810%DMSO). The validity of determining viability by means of measuring cell esterase activity by 2D-FS using fluorescein diacetate as a fluorogenic substrate was verified by comparison with microscopic evaluation of fluorescein fluorescence as well as with the routinely adopted trypan blue exclusion test.