Imaging mass cytometry (IMC) combines the principles of flow cytometry and mass spectrometry (MS) with laser scanning spatial resolution and offers unique advantages for the analysis of tissue samples in unprecedented detail. In contrast to conventional immunohistochemistry, which is limited in its application by the number of possible fluorochrome combinations, IMC uses isoptope-coupled antibodies that allow multiplex analysis of up to 40 markers in the same tissue section simultaneously. In this report we use IMC to analyze formalin-fixed, paraffin-embedded conjunctival tissue. We performed a 18-biomarkers IMC analysis of conjunctival tissue to determine and summarize the possibilities, relevance and limitations of IMC for deciphering the biology and pathology of ocular diseases. Without modifying the manufacturer's protocol, we observed positive and plausible staining for 12 of 18 biomarkers. Subsequent bioinformatical single-cell analysis and phenograph clustering identified 24 different cellular clusters with distinct expression profiles with respect to the markers used. IMC enables highly multiplexed imaging of ocular samples at subcellular resolution. IMC is an innovative and feasible method, providing new insights into ocular disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.