High quality experimental data for isotopic compositions in irradiated fuel are important to spent fuel applications, including nuclear safeguards, spent fuel storage, transportation, and final disposal. The importance of these data has been increasingly recognized in recent years, particularly as countries like Finland and Sweden plan to open the world’s first two spent fuel geological repositories in 2020s, while other countries, including the United States, are considering extended dry fuel storage options. Destructive and nondestructive measurements of a spent fuel rod segment from a Combustion Engineering 14 × 14 fuel assembly of the Calvert Cliffs Unit 1 nuclear reactor have been recently performed at Oak Ridge National Laboratory (ORNL). These ORNL measurements included two samples selected from adjacent axial locations of a fuel rod with initial enrichment of 3.038 wt% 235U, which achieved burnups close to 43.5 GWd/MTU. More than 50 different isotopes of 16 elements were measured using high precision measurement methods. Various investigations have assessed the quality of the new ORNL measurement data, including comparison to previous measurements and to calculation results. Previous measurement data for samples from the same fuel rod measured at ORNL are available from experiments performed at Pacific Northwest National Laboratory in the United States and the Khoplin Radium Institute in Russia. Detailed assembly models were developed using the newly released SCALE 6.2 code package to simulate depletion and decay of the measured fuel samples. Results from this work show that the new ORNL measurements provide a good quality radiochemical assay data set for spent fuel with relatively high burnup and long cooling time, and they can serve as good benchmark data for nuclear burnup code validation and spent fuel studies.