AbstractSegmentation of medical images into different tissue types is essential for many advancements in orthopaedic research; however, manual segmentation techniques can be time- and cost-prohibitive. The purpose of this work was to develop a semi-automatic segmentation algorithm that leverages gradients in spatial intensity to isolate the patella bone from magnetic resonance (MR) images of the knee that does not require a training set. The developed algorithm was validated in a sample of four human participants (in vivo) and three porcine stifle joints (ex vivo) using both magnetic resonance imaging (MRI) and computed tomography (CT). We assessed the repeatability (expressed as mean ± standard deviation) of the semi-automatic segmentation technique on: (1) the same MRI scan twice (Dice similarity coefficient = 0.988 ± 0.002; surface distance = − 0.01 ± 0.001 mm), (2) the scan/re-scan repeatability of the segmentation technique (surface distance = − 0.02 ± 0.03 mm), (3) how the semi-automatic segmentation technique compared to manual MRI segmentation (surface distance = − 0.02 ± 0.08 mm), and (4) how the semi-automatic segmentation technique compared when applied to both MRI and CT images of the same specimens (surface distance = − 0.02 ± 0.06 mm). Mean surface distances perpendicular to the cartilage surface were computed between pairs of patellar bone models. Critically, the semi-automatic segmentation algorithm developed in this work reduced segmentation time by approximately 75%. This method is promising for improving research throughput and potentially for use in generating training data for deep learning algorithms.