Transparent exopolymer particles (TEPs) and their precursors play a critical role in various environmental processes, including contaminant transport and membrane fouling. This study presents an improved Alcian blue (AB) staining-based TEP measurement method and investigates spectroscopic proxies to enhance analysis convenience and reproducibility. The proposed method employs deionized (DI) water and sonication to extract particulate TEPs (TEP0.4μm) and particulate TEPs and their precursors (TEP10kDa), thus eliminating the need for concentrated sulfuric acid and simplifying the procedure with an integrated standard curve. This AB-DI method had a low detection limit of 5 μg Xeq/L for a 1 L sample volume, allowing precise measurements to be made across a broad concentration range. When used in wastewater treatment and microfiltration membrane filtrates, the AB-DI method provided insights into the removal mechanisms of TEPs and their precursors. This study also identified effective spectroscopic proxies by correlating TEP concentrations with optical properties, achieving remarkable estimation accuracy using the UV254 absorbance of TEP-extracted solutions. The UV254 and fluorescence intensities of the protein-like peaks in raw water samples demonstrated significant potential as proxies for TEPs and their precursors. The proposed AB-DI method represents a viable approach for the real-time TEP monitoring for desalination and water reuse processes.