The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1 Ab) and secondary (2 Ab) antibody pair. Although 1 Abs from different species have been used with differently labeled species-specific 2 Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1 Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1 Ab/2 Ab complex and therefore to prevent the cross-reactivity of anti-species 2 Abs used in subsequent detection cycles. We present here a fully automated 1 Ab/2 Ab complex heat deactivation (HD) method on Ventana’s BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity,(2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1 Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1 Abs from same species and the corresponding anti-species 2 Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.