<p>Many liver diseases have a distinct zonation pattern. Similarly, most metabolic processes in the hepatic lobule are also spatially organized. Understanding the interplay between a zonated disease pattern and its impact on zonated metabolic liver function requires the joint quantification of both phenomena. Our study presents an image analysis workflow for the joint zonated quantification of multiple parameters from whole-slide images of conventionally stained serial sections of mouse livers. As a proof of concept, we used small stacks of six adjacent sections, differentially stained with HE, GS and four different CYP enzymes from three mice with different severities of steatosis. Portal fields and central veins were annotated and transferred to adjacent slide images via image registration. The result was visually confirmed to avoid errors in the non-rigid transformation process. This approach allowed identifying the same lobules and zones in multiple consecutive sections. Zones were obtained by arbitrarily dividing the distance between portal fields and central veins into twelve intralobular zones. Zonal distributions of various parameter combinations quantified from multiple slides were visualized in the geometry of lobules and zones and plotted as scatter diagrams. Using this workflow, we could visualize differences in the heterogeneous expression patterns of marker proteins in normal and steatotic livers. As a next step, the algorithm presented here can be applied to a scientific question such as quantifying the impact of zonated steatosis of different severity on the zonated expression pattern and the resulting metabolic function of CYP enzymes.</p>