This ELISA is designed, developed and produced for the quantitative measurement of Folate in human plasma or serum samples. Assay calibrators and patient samples are added directly to wells of a microplate that is coated with a Folate-binding protein, along with HRP-conjugated Folate. After the first incubation period, the binding protein on the wall of the microtiter well captures sample Folate and HRP-conjugated Folate in a competitive nature. Excessive amounts of unbound proteins and HRP-conjugated Folate in each microtiter well are washed away. For the detection of the captured sample Folate, the well is incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the complex bound to the Folate on the wall of the microtiter well is directly proportional to the amount of Folate in the sample. A calibrator standard curve is generated by plotting the absorbance versus the Folate concentration for each calibrator on point-to-point or cubical scales or 4 parameter curve fits. The concentration of Folate in test samples is determined directly from this standard curve.