The efficient delivery of therapeutic drugs, vaccines, peptides, nucleic acids and nanoparticles into cells and tissues is of growing importance to medicine and the pharmaceutical industry. However, the efficacy of uptake and the specific cellular pathways and organelles required for drug delivery into cells are still poorly understood. Using live cell, 3D spinning disk fluorescence microscopy and rapid 3D stacked imaging reconstructs, we tracked intracellular routes and full-cell distributions during the uptake of peptides in macrophages using a soluble, fluorescently-tagged peptide based on a bioactive, plant cyclic peptide, SFTI-1 (de Veer et al, 2021). Alexa-SFTI-1 was compared to the uptake of other cargo - peptides with varying solubility and surface-bound ovalbumin - and endocytic compartments were defined with fluorescent Rab GTPases protein and other markers. In contrast to other methodologies, our approach using 3D light confocal microscopy image stacks analysed by custom automated scripts and machine-learning segmentation algorithms, records the entire volume and number of vesicles in individual cells, in large sample sizes (> 3000 cells/condition) for multiparametric and statistical outputs. In untreated RAW264.7 macrophages, we find that Alexa-SFTI-1 is exclusively internalised by fluid-phase macropinocytosis, verified by inhibitor ablation, at a rate of 8.3 µm3 (femtoliter)/cell in 15 min, in macropinosomes averaging 1.4µm in diameter. Through macropinosome fission and fusion, captured in 3D-reconstructed movies, the peptide is transferred to Rab5-endosomes. After 1 hour of incubation, on average, 70% of ingested Alexa-SFTI-1 is found in Rab7-labelled endo-lysosomes, with fates thereafter including unexpected expulsion of soluble cargo at the cell surface from Rab7-tubules. Since inflammation, infection and other disease-associated physiologies affect drug delivery, we can also investigate these effects. For instance, in LPS-induced proinflammatory macrophages, Alexa-SFTI-1 uptake occurs through enlarged macropinosomes (averaging 1.82µm in diameter) and additionally via induced small vesicle pathways, which together double peptide uptake 17.6 µm3/cell in 15 min. Thus, our novel 3D imaging assay generates high-resolution, comprehensive, volumetric data that enables statistical measurement of peptide and drug uptake on a per-cell basis, alongside tracking organelles and fates for the ingested cargo, including revealing new trafficking events. This approach enhances preclinical testing of therapeutics and nanoparticles to inform dosing and fates in different cell types and under different physiological or genetic conditions and it is currently being adapted for use in tissue environments.