The time-resolved fluorescence technique, quenching resonance energy transfer, QRET, relies on a single-labeled binding partner in combination with a soluble quencher. The quencher reduces efficiently the fluorescence of the unbound labeled ligand, whereas the fluorescence of the bound fraction is detectable. This approach allows the development of homogeneous screening assays in a simple and cost-effective manner. In this study, two single-label fluorometric methods, fluorescence polarization (FP) and the QRET technique, are compared in a simple biochemical model immunoassay of estradiol. Estradiol-6-amino was labeled with fluorescein and lanthanide(III) chelates for the FP and QRET assays, respectively. The labeled estradiols were allowed to compete against free estradiol, and the assay parameters were investigated. The EC(50) value of QRET assay using europium(III)-labeled estradiol was 0.1 nM, and the assay sensitivity was approximately 10 pM. These values were more than 10-fold lower than those for the FP assay with Z' values higher than 0.75 for both assays. The high sensitivity was attributed to a low concentration of antibody fragment and labeled estradiol used in the QRET assay. This reduces cost in screening studies without sacrificing the assay performance. A signal-to-background ratio (S/B) of more than 20 was reached in the QRET assay at elevated concentration of the assay components, whereas S/B of 3.6 was measured in the FP assay when both assays shared the same EC(50) value of 1 nM. Multiplexing of assays is a cost-effective means to run screening studies as multiple data can be extracted from a single experiment. Therefore, multiplexing of the QRET assay was investigated and its feasibility was successfully demonstrated in a dual-parametric assay using estradiol labeled with europium(III) and terbium(III) chelates.