We developed a multiplexed point-of-care immunodiagnostic assay for antibody detection in human sera made through the vertical stacking of functional paper layers. In this multiplexed vertical flow immunodiagnostic assay (xVFA), a colorimetric signal is generated by gold nanoparticles captured on a spatially-multiplexed sensing membrane containing specific antigens. The assay is completed in 20 minutes, following which the sensing membrane is imaged by a cost-effective mobile-phone reader. The images are sent to a server, where the results are rapidly analyzed and relayed back to the user. The performance of the assay was evaluated by measuring Lyme-specific antibodies in human sera as model target antibodies. The presented platform is rapid, simple, inexpensive, and allows for simultaneous and quantitative measurement of multiple antibodies and/or antigens making it a suitable point-of-care platform for disease diagnostics.