The enzyme-linked immunosorbent assay (ELISA) is an immunological biochemical assay used to detect and quantify proteins in various sample types including serum, plasma, cell culture medium, cleared bacterial lysate, and buffer. To detect the antigen of interest in these samples, specific antibodies targeting accessible epitopes on antigen’s surface are employed. Highly affine antibodies with excellent specificity enable accurate and precise detection and quantification of the protein of interest. This high affinity also facilitates the detection of trace amounts of proteins, which is crucial for analyzing cytokines typically present in much lower concentrations compared to plasma proteins. Plasma levels of oxidized macrophage migration inhibitory factor (oxMIF) can reach 150 ng/mL or higher, but concentrations below 20 ng/mL are more common. In addition, differentiating between the pathological isoform oxMIF and MIF is necessary. Here, we describe a method to detect oxMIF and MIF in various sample matrices of biological origin using the sandwich ELISA method.