Abstract Background and aims: The reference method for diagnosing porto-systemic shunting (PSS) in experimental portal hypertension involves measuring (51)Chrome ((51)Cr)-labelled microspheres. Unfortunately, this technique necessitates the sacrifice of animals. Alternatively, (99m)technetium-macroaggregated albumin ((99m)Tc-MAA) has been used; however, planar scintigraphy imaging techniques are not quantitatively accurate and adequate spatial information is not attained. Here, we describe a reliable, minimally invasive and rapid in vivo imaging technique, using three-dimensional single photon emission computed tomography (3D SPECT) modus, that allows more accurate quantification, serial measurements and spatial discrimination. Methodology: Partial portal vein ligation, common bile duct ligation and sham were induced in male mice. A mixture of (51)Cr microspheres and (99m)Tc-macroaggregated albumin particles was injected into the splenic pulpa. All mice were scanned in vivo with muSPECT (1 mm spatial resolution) and, when mandatory for localisation, a muSPECT-CT was acquired. A relative quantitative analysis was performed based on the 3D reconstructed datasets. Additionally, (51)Cr was measured in the same animals to calculate the correlation coefficient between the (99m)Tc detection and the gold standard (51)Cr. In each measuring modality, the PSS fraction was calculated using the formula: [(lung counts)/(lung counts+liver counts)] x 100. Results: A significant correlation between the (99m)Tc detection and (51)Cr was demonstrated in partial portal vein ligation, common bile duct ligation and sham mice and there was a good agreement between the two modalities. muSPECT scanning delivers high spatial resolution and 3D image reconstructions. Conclusion: We have demonstrated that quantitative high-resolution muSPECT imaging with (99m)Tc-MAA is useful for detecting the extent of PSS in a non-sacrificing set-up. This technology permits serial measurements and high-throughput screening to detect baseline PSS, which is especially important in pharmacological studies.