A method to determine the neutron production rate of a spent nuclear fuel segment sample by means of non-destructive assay conducted under standard controlled-area conditions is described and demonstrated. A neutron well counter designed for routine nuclear safeguards applications is applied. The method relies on a transfer procedure that is adapted to the hot cell facilities at the Laboratory for High and Medium level Activity of SCK CEN in Belgium. Experiments with 252Cf(sf) sources, certified for their neutron emission rate, were carried out at the Joint Research Centre to determine the characteristics of the detection device. Measurements of a segment of a spent nuclear fuel rod were carried out at SCK CEN resulting in an absolute and non-destructive measurement of the neutron production rate avoiding any reference to a representative spent nuclear fuel sample to calibrate the device. Results of these measurements were used to study the performance of depletion codes, i.e., ALEPH2, SCALE, and Serpent2. The study includes a code-to-code and code-to-experiment comparison using different nuclear data libraries.