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Three-dimensional (3D) imaging has a significant impact on many challenges of life sciences. Three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging label-free bioanalytical technique capturing the spatial distribution of hundreds of molecular compounds in 3D by providing a MALDI mass spectrum for each spatial point of a 3D sample. Currently, 3D MALDI-IMS cannot tap its full potential due to the lack efficient computational methods for constructing, processing, and visualizing large and complex 3D MALDI-IMS data. We present a new pipeline of efficient computational methods, which enables analysis and interpretation of a 3D MALDI-IMS data set. Construction of a MALDI-IMS data set was done according to the state-of-the-art protocols and involved sample preparation, spectra acquisition, spectra preprocessing, and registration of serial sections. For analysis and interpretation of 3D MALDI-IMS data, we applied the spatial segmentation approach which is well-accepted in analysis of two-dimensional (2D) MALDI-IMS data. In line with 2D data analysis, we used edge-preserving 3D image denoising prior to segmentation to reduce strong and chaotic spectrum-to-spectrum variation. For segmentation, we used an efficient clustering method, called bisecting k-means, which is optimized for hierarchical clustering of a large 3D MALDI-IMS data set. Using the proposed pipeline, we analyzed a central part of a mouse kidney using 33 serial sections of 3.5 μm thickness after the PAXgene tissue fixation and paraffin embedding. For each serial section, a 2D MALDI-IMS data set was acquired following the standard protocols with the high spatial resolution of 50 μm. Altogether, 512 495 mass spectra were acquired that corresponds to approximately 50 gigabytes of data. After registration of serial sections into a 3D data set, our computational pipeline allowed us to reveal the 3D kidney anatomical structure based on mass spectrometry data only. Finally, automated analysis discovered molecular masses colocalized with major anatomical regions. In the same way, the proposed pipeline can be used for analysis and interpretation of any 3D MALDI-IMS data set in particular of pathological cases.