Phosphorylation and dephosphorylation play pivotal roles in cellular regulation and signaling processes. [1,2] Numerous inhibitors have been proposed or applied to regulate the process of phosphorylation/dephosphorylation. [3] A sensitive assay to report change of the phosphorylation state will be extremely valuable for biomedical applications. As the chemical difference in the process is only a phosphate group, developing a simple and sensitive assay is challenging. Herein we present a novel design of a gold-nanoparticlebased colorimetric assay to detect phosphatase activity. Gold nanoparticles (Au-NP) have interesting optical and electronic properties, that is, a strong surface plasmon resonance at around 520 nm in aqueous solutions, which is attributed to collective oscillations of surface electrons induced by incoming visible light. [4,5] The exact plasmon resonance band is dependant on the size, shape, surrounding medium, and distance of the particles. [6][7][8] Upon aggregation of Au-NP, a significant red shift in absorbance is observed. This colorimetric nano-gold-aggregation (NGA) phenomenon has been applied to several assays, such as solution-based immunoassays, [9] DNA-hybridization assay, [10] and glucose-sensing. [11] To form NGA, binding between two molecules, for example, antigen-antibody, complimentary oligonucleotides, or guesthost complexes, is needed. Recently, a new design to detect enzymatic activity by using the NGA phenomenon but not utilizing binding has been reported. [12] In that design, acetylated cysteine residues were added to both termini of the peptide substrate. As the cysteine could bind to Au-NP effectively, the intact peptide substrates cross-linked Au-NP through the two cysteine arms, resulting in colorimetric changes. Conversely, the proteolyzed peptide fragments, with only one cysteine residue, could not bridge Au-NP, resulting in no colorimetric change. Based on this NGA-induced absorption change, protease activity can be conveniently determined by measuring the reduction of aggregation. Recently, Levy et al. reported that not only the thiol group but also positively charged amine groups that have a strong affinity for gold. [13] Short peptides containing a cysteine and a lysine or an arginine residue at both ends of the peptide were