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Two new protein conjugates were prepared and studied to develop and compare two (direct and indirect) competitive enzyme-linked immunosorbent assay (ELISA) formats for the determination of cortisol in human saliva. Toward this goal, ovalbumin was conjugated to cortisol and used for developing an indirect competitive ELISA, while alkaline phosphatase was coupled with the same analyte for a direct competitive assay. The yield of the conjugation reactions was evaluated. The results obtained show that the indirect and direct ELISA formats developed for cortisol had working ranges of 0.5–70 and 2–330ng/ml and detection limits of 0.5 and 1.2ng/ml, respectively. Artificial and real saliva samples were spiked with cortisol to study the matrix effect of saliva. The suitability of the assays for quantification of cortisol in saliva was also studied.