A simple, label-free cytometry technique is introduced. It is based on the analysis of the fluctuation of image Gray Level Information Entropy (GLIE) which is shown to reflect intracellular biophysical properties like generalized entropy. In this study, the analytical relations between cellular thermodynamic generalized entropy and diffusivity and GLIE fluctuation measures are explored for the first time. The standard deviation (SD) of GLIE is shown by experiments, simulation and theoretical analysis to be indifferent to microscope system “noise”. Then, the ability of GLIE fluctuation measures to reflect basic cellular entropy conditions of early death and malignancy is demonstrated in a cell model of human, healthy-donor lymphocytes, malignant Jurkat cells, as well as dead lymphocytes and Jurkat cells. Utilization of GLIE-based fluctuation measures seems to have the advantage of displaying biophysical characterization of the tested cells, like diffusivity and entropy, in a novel, unique, simple and illustrative way.