A protocol for measuring the total plasma homocysteine (tHCY) concentration in very small samples using a selective, recombinant homocysteine α, γ-lyase (rHCYase) and a small portable fluorescence reader is described. The rHCYase produces hydrogen sulfide (H₂S) from tHCY without interference from physiological concentrations of Cys or other plasma components. H₂S is determined through reactivity with N,N-dibutyl phenylene diamine (DBPDA), which leads to the formation of a fluorescent chromophore. Only 5 μl of plasma/serum sample is required, which can be obtained from a finger prick, suggesting great potential for mass screening. The assay takes ∼20 min.