Vitamin D is necessary for the healthy growth and development of bone and muscle. Vitamin D deficiency， which is present in 42% of the US population， is often undiagnosed as symptoms may not manifest for several years and long-term deficiency has been linked to osteoporosis， diabetes and cancer. Currently the majority of vitamin D testing is performed in large-scale commercial laboratories which have high operational costs and long times-to-result. Development of a low-cost point-of-need assay could be transformative to deficiency analysis in limited-resource settings. The best biomarker of vitamin D status， 25hydroxyvitamin D (25(OH)D)， however， is particularly challenging to measure in such a format due to complexities involved in sample preparation， including the need to separate the marker from its binding protein. Here we present a rapid diagnostic test for the accurate， quantitative assessment of 25(OH)D in finger-stick blood. The assay is accompanied by a smartphone-assisted portable imaging device that can autonomously perform the necessary image processing. To achieve accurate quantification of 25(OH)D， we also demonstrate a novel elution buffer that separates 25(OH)D from its binding protein in situ， eliminating the need for sample preparation. In human trials， the accuracy of our platform is 90.5%.