Alhydrogel (aluminum hydroxide) is a widely used adjuvant in the US. Regulatory authorities require that vaccines be tested to determine the antigen content in the final vaccine product. The level of formulated antigen is currently determined in our laboratory by the o-Phthalaldehyde (OPA) fluorescent protein assay, and antigen identity and integrity are determined by Western blot and SDS-PAGE. However, OPA assay is non-specific and only limited to detection of total protein content, and it is often not sensitive enough to detect antigens in low dose formulations. Furthermore, antigens used in identity and integrity tests must be extracted from vaccines using an extraction procedure which is time-consuming and may not completely recover antigens for analysis or may alter the structures of antigens during extraction. The present study developed a Direct Alum Formulation Immunoassay (DAFIA) which was designed to directly (without antigen extraction), accurately, and sensitively determine the antigen content, identity and integrity on alum. The AMA1-C1/Alhydrogel formulation was used as a model vaccine in assay development and validation. The results showed that the DAFIA is highly antigen-specific, accurate (87-100%), sensitive (0.16 microg/ml), reproducible, and simple with a linear detection range of 0.16-10 microg/ml. These results demonstrate that DAFIA is an excellent assay to determine antigen content, identity and integrity of antigens bound to alum and may be used in routine vaccine quality control for testing antigens in Alhydrogel-based vaccines.