This ELISA (enzyme-linked immunosorbent assay) kit is intended for the quantitative determination of human pepsinogen I levels in serum. Determination of human serumpepsinogen I level would be a useful tool in the aid of diagnosing the functional states of acid-secreting gastric mucosa. This ELISA is designed, developed and produced for the quantitative measurement of human pepsinogen I level in serum sample. The assay utilizes the two-site “sandwich” technique with two selected monoclonal antibodies that bind to different epitopes of human pepsinogen I without any cross-reaction to human pepsinogen II. Assay standards,controls and patient serum samples containing human pepsinogen I are added directly to microtiter wells of microplate that was coated with streptavidin. Simultaneously, a biotinylated antibody and a horseradish peroxidase-conjugated antibody are added to each well. After the first incubation period, the wall of microtiter well captures the biotinylated antibody as well as an immuno complex in the form of “streptavidin – biotin-antibody – pepsinogen I – HRP-antibody”. Unbound proteins as well as unbound HRP-conjugated antibody in each microtiter well are removed in the subsequent washing step. 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 tracer antibody bound to the pepsinogen I on the wall of the microtiter well is directly proportional to the amount of pepsinogen I in the sample. A standard curve is generated by plotting the absorbance versus the respective human pepsinogen I concentration for each standard on Point-to-Point, Cubic Spline or 4-Parameter plot. The concentration of human pepsinogen I in test samples is determined directly from this standard curve.