Vitamin D deficiency has been linked to several diseases and adverse outcomes including: diabetes, cardiovascular diseases, and even cancer. At present the clear majority of vitamin D testing is performed in large-scale laboratories at the request of a physician as part of an annual panel of blood tests. Here we present a paper-based microfluidic system for rapid quantification of vitamin D levels on a portable platform. The system consists of a smartphone accessory, an app, and a test strip that allows the colorimetric detection of 25-hydroxyvitamin D using a novel gold nanoparticle-based immunoassay. We show that the system can be used to accurately measure physiological levels of 25-hydroxyvitamin D with accuracy better than 25 nM and a precision of 10 nM. We compare our system with well-established ELISA test kits for serum samples of unknown concentration and demonstrate equivalency of the results.