Multiplex immunofluorescence (mIF) has arisen as an important tool for immuno-profiling tumor tissues. We updated our manual protocol into an automated protocol that allows the use of up to seven markers in five mIF panels to apply to formalin-fixed paraffin-embedded tumor tissues. Using a tyramide signal amplification system, we optimized five mIF panels that included cytokeratin to characterize malignant cells (MCs), immune checkpoint markers (i.e., PD-L1, B7-H3, B7-H4, IDO-1, VISTA, LAG3, ICOS, TIM3, and OX40), tumor-infiltrating lymphocytic markers (i.e., CD3, CD8, CD45RO, granzyme B, PD-1, and FOXP3), and markers to characterize myeloid-derived suppressor cells (i.e., CD68, CD66b, CD14, CD33, Arg-1, and CD11b). To determine analytical reproducibility and the impact of those panels for immuno-profiling tumor tissues, we performed an exploratory analysis in a set of non–small cell lung cancer (NSCLC) samples. The slides were scanned, and the different cell phenotypes were quantified by simultaneous co-localizations with the markers using image analysis software. Comparison between the time points of staining showed high analytical reproducibility. The analysis of NSCLC cases showed an immunosuppressive microenvironment with PD-L1/PD-1 expression as a predominant axis. Interestingly, high density of MCs expressing B7-H4 was correlated with recurrence. Unexpectedly, MCs expressing OX40 were also detected, and those cells were a closer distance to CD3+T-cells than were MCs expressing other immune checkpoints. Two different cellular patterns of spatial distribution were determined according the CD3 distribution, and the predominant pattern was related with active immunosuppressive interaction with MCs. Our study shows that these five mIF panels can identify multiple targets in a single cell with high reproducibility. The study of different cell populations and their spatial relationship can open new ideas for therapeutic approaches.