A membrane-based assay device for detecting the presence or quantity of an analyte residing in a test sample is provided. The device utilizes a self-calibrated magnetic binding assay format (e.g., sandwich, competitive, etc.) that includes detection probes capable of generating a detection signal (e.g., fluorescent non-magnetic particles) and calibration probes capable of generating a calibration signal (e.g., fluorescent magnetic particles). The amount of the analyte within the test sample is proportional (e.g., directly or inversely) to the intensity of the detection signal calibrated by the intensity of the calibration signal. It has been discovered that the fluidics-based device of the present invention provides an accurate, inexpensive, and readily controllable method of determining the presence of an analyte in a test sample.