Probe-based Spatial Transcriptomics profiles spatially-resolved transcriptomes using gene-specific probe pairs for both Formalin-fixed paraffin-embedded (FFPE) and Fresh Frozen samples. However, its applicability is restricted to human and mouse studies, due to commercial probe set availability. Here, we present ProbeST, an open-source computational pipeline for designing custom probe sets for genes of interest of a given organism. We validated ProbeST on FFPE mouse enteroid-derived monolayers infected with Salmonella enterica serovar Typhimurium, using custom pathogen probes with the available mouse probe panel. We simultaneously detected host and pathogen transcripts, with high probe specificity and low sensitivity against mCherry imaging, enabling identification of inflammatory response host genes Mefv, Tnf, and Anxa1 colocalizing to pathogen genes. The reproducible ProbeST workflow expands probe-based Spatial Transcriptomics to studies of non-model organisms and host–pathogen interactions.