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Dual-Energy computed tomography (DECT) is expected to allow for more accurate proton therapy treatment planning by improving the estimation of relative stopping power (SPR) and other tissue properties.In this study, we investigated the accuracy of SPR prediction and tissue segmentation based on dual- and Single-Energy CT (SECT). For this purpose, fresh animal tissue samples were irradiated in a clinical proton therapy facility and high spatial-resolution three-dimensional proton dose distributions were obtained using dosimetric polymer gel downstream to the samples. The accuracy of this setup was benchmarked against depth-dose measurements obtained with an ionization chamber behind an adjustable water column (peakfinder, PTW, Germany). The predicted SPR values showed good consistency for both methods with deviations below 1%.DECT (90/150 kVp) and SECT (120kVp) images were acquired and converted to SPR and tissue compositions as input for MC- simulations. DECT-to-SPR conversion yielded mean errors of 0.5%, outperforming the SECT calibration with 1.1% deviation from peakfinder results.A detailed comparison of measured dose distributions to MC-simulations for highly inhomogeneous samples will be presented.In summary, dosimetric gel was used to obtain 3D high-resolution proton ranges and compared to peakfinder measurements and MC-simulations, in order to quantify the accuracy of DECT and SECT based tissue characterization.