PyNutil is a Python library for brain-wide quantification and spatial analysis of features in serial section images from mouse and rat brain. It aims to replicate the Quantifier feature of the Nutil software (RRID: SCR_017183) to be run locally or to be intergrated with the QUINT workflow web-tools. It builds on registration to a standardised reference atlas with the QuickNII (RRID:SCR_016854) and VisuAlign software (RRID:SCR_017978) and feature extraction by segmentation with an image analysis software such as ilastik (RRID:SCR_015246).