Small-molecule neurotransmitters, their precursors and metabolites are involved in the brain chemical network and transmit signals between neurons. At present, researchers rely on indirect histochemical, immunohistochemical, and ligand-based assays to detect these small-molecule neurotransmitter substances or direct analysis of tissue homogenates using HPLC or LC-MS/MS. Current neuroimaging techniques have very limited abilities to directly identify and quantify neurotransmitters from brain sections. By performing MALDI mass spectrometry imaging (MSI) directly on the surface of a tissue section, the technique has quickly been established as a powerful in situ visualization tool for measuring abundance and spatial distribution of endogenous and pharmaceutical compounds, lipids, peptides and small proteins. We recently introduced a reactive MALDI matrix, which selectively targets the primary amine group of neurotransmitters, metabolites and neuroactive substances but also function as a matrix for the ionization (1,2). However, the limitation of using such reactive matrix is its limitation to target all downstream dopamine metabolites derived from monoamine oxidase (MAO) or catechol-O-methyltransferase (COMT) enzymes. The majority of small molecule neurotransmitters such as catecholamines, amino acids and trace amines possess phenolic hydroxyl and/or primary or secondary amines, which are proper nucleophilic groups. We therefore developed a new reactive matrix that can selectively target and charge-tag both phenolic and primary amine groups, thus enabling MALDI-MSI of both MAO and COMT downstream metabolites. By this developed reactive matrix, we were able to detect and map the localization of most of the neurotransmitters and metabolites involved in the dopaminergic and serotonergic network in a single brain tissue section (e.g., in Parkinson's disease) and it represents a novel methodology, which assists their identification through the selectivity of the reaction. The sensitivity and specificity of the imaging approach of neurochemicals has a great potential in many diverse applications in fields such as neuroscience, pharmacology, drug discovery, neurochemistry, and medicine.