The xanthophyll carotenoids lutein and zeaxanthin constitute the major carotenoids of the macular pigment in the human retina where they are thought to act in part to prevent light induced oxidative damage associated with age-related macular degeneration (AMD). The highly selective uptake of these pigments is mediated by specific carotenoid-binding proteins (GSTP1 and StARD3) recently identified in our laboratory. Carotenoids are hydrophobic in nature, so we first systematically optimized carotenoid preparations that are nano-dispersed in aqueous buffers, and then we used a new-generation surface plasmon resonance (SPR) protocol called FastStep™, which is significantly faster than conventional SPR assays. We have explored carotenoid-binding interactions of five proteins: human serum albumin (HSA), β-lactoglobulin (LG), steroidogenic acute regulatory domain proteins (StARD1, StARD3) and glutathione S- transferase Pi isoform (GSTP1). HSA and LG showed relatively weak interaction with carotenoids (K(D)>1 μM). GSTP1 evidenced high affinity and specificity towards zeaxanthin and meso-zeaxanthin with K(D) values 0.14±0.02 μM and 0.17±0.02 μM, respectively. StARD3 expressed a relative high specificity towards lutein with a K(D) value of 0.59±0.03 μM, whereas StARD1 exhibited a relatively low selectivity and affinity (K(D)>1 μM) towards the various carotenoids tested.