Pyridoxal-5′-phosphate (PLP) is the biologically active form of vitamin B₆. Clinical studies suggest that low PLP concentrations are an independent risk factor for cardiovascular and other diseases. However, PLP concentrations are not routinely used for diagnosis because of the lack of a homogeneous, nonradioactive assay. We describe a homogeneous, nonradioactive, enzymatic PLP assay that uses the apo form of the recombinant PLP-dependent enzyme, homocysteine-α,γ-lyase (rHCYase). The restoration of enzymatic activity by reconstitution of the holoenzyme is linearly dependant on the amount of PLP bound to the enzyme. Nanomolar concentrations of PLP can then be measured by the conversion (by reconstituted holo-rHCYase) of millimolar concentrations of homocysteine (HCY) to H₂S. H₂S combines with DBPDA (N,N-dibutylphenylenediamine) to form 3,7-Bis(dibutylamino)-phenothiazine-5′-ium chloride and the absorbance of this compound is read at 675 nm. The PLP enzymatic assay has a lower limit of detection of 14.8 nmol l−1 and is linear to 300 nmol l−1 and requires only 10 μl plasma or serum. This PLP assay is the first homogeneous, nonradioactive method for diagnosing vitamin B₆. The procedure takes ∼2 h to complete.