Hormones are important bioactive compounds in blood and tissue that vary in concentration in response to stress and certain disease states. Establishing the changes in physiological hormone concentrations over time can lead to more effective diagnoses and perhaps a better understanding of the evolution of stress and disease. To monitor concentration over time, the sampling must be rapid and noninvasive; specimens such as saliva that require little effort to collect are preferred. However, more sensitive assay techniques are needed when compared to blood analysis since free hormone concentration in saliva is only a small fraction of the concentration in circulating blood. In this work, magnetic field-induced structures of paramagnetic particles are used as a solid substrate to demonstrate improved detection limits for a separation-free assay of cortisol. Once formed, the structures are subjected to a rotating magnetic field and this leads to two important features. First is the ability to utilize frequency and phase filtering (lock-in amplification) for the signal generated from surface-bound labeled species. Second is the improved mass transport of the antigen to the surface of the rotating structures. These two unique capabilities result in a quantifiable signal at a relatively low target antigen concentration. This method has been demonstrated with the detection of fluorescein isothiocyanate-labeled cortisol (FITC-cortisol) at a concentration of 300 pM.