Sol–gel encapsulation has been used as the basis for detecting cortisol by an immunoassay approach. Previous research showed that antibodies immobilized in the pores of a sol–gel derived silica were able to bind cortisol and be used as an immunosensor. However, this approach was not effective when measuring cortisol levels in human serum because of interference from other fluorescence sources. The present paper describes a protocol which overcomes these limitations and enables sol–gel immunoassays to effectively measure cortisol in human serum over the physiological range of cortisol blood concentrations in an adult (2–28 μg/dL). The method involves a standard additions approach in which various amounts of cortisol are added to the serum. The cortisol concentration values obtained with our sol–gel immunoassay were typically within 10% of the values obtained by traditional analytical methods. The protocol presented here represents a significant contribution to sol–gel sensing and immunoassays in particular, because of the ability to detect an analyte in human serum. In addition, this work reports the first comparison between results from a sol–gel immunosensor and an alternative immuno-binding method for analyte detection.