Introduction: Laser Interstitial Thermal Therapy (LITT) is a minimally invasive, hyperthermia-based laser cytoreductive method used in the treatment of several severe conditions, including inoperable brain tumors, treatment-resistant epilepsy, and radiation necrosis. Laser-induced heat causes damage to the tissue and alters the brain-tissue microenvironment (TME) at the local treatment site and beyond. The regional effects include activation of resident brain cells, release of mediators of inflammation, and immune cell infiltration, which may activate immune responses. Immunomodulatory responses to LITT in the brain microenvironment are largely unknown. One hurdle is the lack of affordable and efficient imaging protocols that enable the simultaneous spatial visualization of multiple immunoreactive cellular targets in the brain. Here, we describe a flexible and affordable multiplex immunofluorescence (mIF) staining method in our established LITT mouse model. In the post-LITT mouse brain, mIF revealed laser ablation-induced complex spatial responses by the resident brain and innate immune cells. Methods: We developed a customizable and affordable mIF detection protocol that uses standard histological and microscopy equipment to assess TME changes on single sections of formalin-fixed paraffin-embedded (FFPE) mouse brain tissues at day 6 post-LITT. This mIF imaging procedure provided a comprehensive spatial analysis of multiple cellular and molecular immunotargets in resident brain cell responses and immune cell profiles in individual tissue sections, as exemplified with a mouse brain microenvironment on day 6 post-LITT. Results: We optimized mIF imaging methodology on LITT-treated mouse brain FFPE serial sections to visualize the spatial distribution of resident brain cell cellular activation states and the emergence and distribution of immune cell populations at the LITT site. Our mIF imaging protocol of mouse brain regions surrounding the LITT site revealed the activation of resident astrocytes and microglial cells and the presence of a diverse innate immune cell population in this region. Conclusion: The combined use of LITT ablation treatment with multiplexed versus regular immunofluorescence offers a significant advancement and aids in our understanding of post-LITT repair and immune responses in the brain. We characterized the thermal ablation-induced modulation of resident brain cells and innate immune cell infiltrations.