The cellular level of nicotinamide adenine dinucleotide (NAD ⁺ ), through its different functions, affects cellular metabolism and signalling ^1-3 . A decrease in the NAD ⁺ content has been associated with various pathologies and physiological aging ^4,5 , while strategies to boost cellular NAD ⁺ levels have been shown to be effective against age-related diseases in many animal models ⁶ . The link between decreased NAD ⁺ levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD ⁺ , ideally applicable at the point of care. Here, we introduce a bioluminescent biosensor for the rapid quantification of NAD ⁺ levels in biological samples, which can be used either in laboratories or at the point of care. The biosensor is a semisynthetic, light-emitting sensor protein that changes the colour of emitted light from blue to red on binding of NAD ⁺ . This NAD ⁺ -dependent colour change enables the use of the biosensor in paper-based assays in which NAD ⁺ is quantified by measuring the colour of the emitted light by using either a simple digital camera or a plate reader. We used the approach to quantify NAD ⁺ levels in cell culture, tissue and blood samples, yielding results that agreed with those from standard testing methods. The same biosensor furthermore allows the quantification of NAD ⁺ -dependent enzymatic activities in blood samples, thus expanding its utility as a tool for point-of-care diagnostics.