Purpose: It has been proposed that PET images can be used to guide the delivery of selectively escalated doses to biologically‐relevant tumor subvolumes. Histopathological validation of PET imaging is challenging due to difficulties associated with precise registration of non‐invasive in‐vivo images to histopathological ex‐vivo images. The aim of this study is to develop an alternative method of PET imaging validation for image‐guidance applications. The method is applied to evaluation of the feasibility of FDG PET‐based delineation of viable tissue in animal tumor models. Methods: Tumor‐bearing mice were injected with 14C‐FDG. Whole‐tumor specimens were sectioned, obtaining 8μm thick sections every 120μm throughout the tumor. These sections were used to obtain 14C‐FDG autoradiography and H&E microscopy images. Viable tumor tissue was delineated on each H&E image. Based on sequential digital photography images of the tissue block acquired during sectioning, the true 3D distributions of 14C‐FDG and viable tissue were reconstituted. To simulate generation of a PET image, the 3D activity map was convolved with a 3D point‐spread‐function of Siemens Inveon small‐animal PET scanner. Threshold‐based analysis was used to evaluate the degree of coincidence between the areas of high FDG uptake in the simulated PET image and 3D distribution of viable tissue. Results: Averaging effects associated with PET imaging altered the true 3D spatial pattern of FDG intratumoral uptake. ROC analysis indicated good sensitivity of FDG PET image‐segmentation for the detection of the viable tissue (AUC = 0.74). However, the specificity was low, as indicated by the low threshold value at which the maximum overlap occurred (22% of maximum uptake). Conclusion: A novel method of histopathological validation of PET imaging for image‐guidance in radiotherapy was developed. Using this method, it was demonstrated that for the tumors with high viable tissue content, FDG‐thresholding can be used for viable tissue detection.