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The preservation of kidneys using normothermic machine perfusion (NMP) prior to transplantation has the potential for predictive evaluation of organ quality. Investigations concerning the quantitative assessment of physiological tissue parameters and their dependence on organ function lack in this context. In this study, hyperspectral imaging (HSI) in the wavelength range of 500&ndash, 995 nm was conducted for the determination of tissue water content (TWC) in kidneys. The quantitative relationship between spectral data and the reference TWC values was established by partial least squares regression (PLSR). Different preprocessing methods were applied to investigate their influence on predicting the TWC of kidneys. In the full wavelength range, the best models for absorbance and reflectance spectra provided Rp2 values of 0.968 and 0.963, as well as root-mean-square error of prediction (RMSEP) values of 2.016 and 2.155, respectively. Considering an optimal wavelength range (800&ndash, 980 nm), the best model based on reflectance spectra (Rp2 value of 0.941, RMSEP value of 3.202). Finally, the visualization of TWC distribution in all pixels of kidneys&rsquo, HSI image was implemented. The results show the feasibility of HSI for a non-invasively and accurate TWC prediction in kidneys, which could be used in the future to assess the quality of kidneys during the preservation period.