The mesoscale description of the subcellular organization informs about cellular mechanisms in disease state. However, applications of soft X-ray tomography (SXT), an important approach for characterizing organelle organization, are limited by labor-intensive manual segmentation. Here we report a pipeline for automated segmentation and systematic analysis of SXT tomograms. Our approach combines semantic and first-applied instance segmentation to produce separate organelle masks with high Dice and Recall indexes, followed by analysis of organelle localization based on the radial distribution function. We demonstrated this technique by investigating the organization of INS-1E pancreatic β-cell organization under different treatments at multiple time points. Consistent with a previous analysis of a similar dataset, our results revealed the impact of glucose stimulation on the localization and molecular density of insulin vesicles and mitochondria. This pipeline can be extended to SXT tomograms of any cell type to shed light on the subcellular rearrangements under different drug treatments.