Abstract The potential for spatial biology in cancer research is understood and edified. With it we can understand the tumor microenvironment, unravel responsiveness to drug treatments and cell therapies, and ultimately stratify patients for targeted treatment and clinical trial enrollment. Yet, researchers exploring spatial biology may be discouraged by what can be a resource intensive exercise, and most importantly, a commitment of high value patient samples. Furthermore, the design of reagent panels used to study samples is not trivial, and a specimen’s unique biomarker expression profile may require adjustments. Here we present a novel solution that permits sample re-interrogation and in-flight adjustments of reagent panels in our spatial biology workflow. The CellScape™ platform for Precise Spatial Multiplexing paired with VistaPlex™ Assay Kits provide a ready-to-use cyclic multiplex immunofluorescence (IF) solution to interrogate fresh frozen and FFPE tissue samples. Tissues are mounted in enclosed fluidic chambers, while staining and imaging are conducted on the CellScape instrument. The experiment requires no additional autostainer equipment and is divided into separate staining and imaging rounds. The end user may therefore pause between rounds and conduct inter-round analyses, which can then guide decisions of whether and how to continue the experiment. To illustrate the benefit of this approach, we present a use case in which we applied VistaPlex antibody panels to reveal the spatial distribution of major immune cell populations. Inter-round analysis and conclusions were then drawn from this preliminary phenotype map before successive rounds of rationally planned staining were conducted to further deepen the depth and breadth of spatial phenotyping. The CellScape sample reinterrogation feature is a pragmatic spatial biology solution that optimizes the use of precious resources, whilst allowing researchers to generate informed decisions about antibody panel designs almost in real time. This enhanced utility may reduce the resistance to spatial biology among researchers working with samples that are limited or irreplaceable. Citation Format: Matthew H. Ingalls, Xenia Meshik, Charles E. Jackson, Kayla E. Cashion, Arne Christians, Anke Brix, Marcus Boettcher, J Spencer Schwarz, Oliver Braubach. Towards a drag and drop spatial biology workflow: Introducing sample reinterrogation as a conduit for the progressive development and on-line analysis of high parameter spatial biology data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5497.