Objective <p>To validate the automated analysis of magnetic resonance imaging (MRI) diffusion phantoms with an updated version of the magnetic resonance biomarker assessment software (MR-BIAS), an open-source tool initially developed for the analysis of MRI relaxometry phantoms.</p> Materials and methods <p>The updated MR-BIAS was validated against two published diffusion weighted MRI datasets: (i) a single-site study (<i>n</i> = 48) was used for validation of apparent diffusion coefficients (ADC) and to identify optimal region of interest (ROI) selection, and (ii) a multi-centre multi-vendor study including diffusion imaging from a shared benchmark protocol (<i>n</i> = 49) and site-specific protocols (<i>n</i> = 43). ADC analysis compared both datasets with ROIs manually matched to the original studies, and with automatically detected optimal ROIs.</p> Results <p>MR-BIAS ADC values were statistically equivalent (<i>p</i> &lt; 0.05) to original studies within tolerances (manual ROI, automatic ROI) for the single-site study (± 0.01, ± 6 μm²/s) and for the multi-vendor study for benchmark (± 4, ± 7 μm²/s) and site-specific (± 3, ± 6 μm²/s) protocols. The optimal ROI was a central cylinder (height = 10mm, diamete<i>r</i> = 10mm). MR-BIAS ADC summary metrics were comparable to those of the original studies.</p> Discussion <p>MR-BIAS can automatically and accurately perform ADC analysis of diffusion phantoms, making the software suitable for the quality assurance of multi-centre studies of multi-parametric MRI.</p>