<p>The surface chemistry of catalyst nanoparticles is crucial for understanding catalytic mechanisms of reactions significant for chemical transformation, energy conversion and environmental sustainability. To enable a high-vacuum X-ray photoelectron spectroscopy (XPS) system to characterize nanoparticle surfaces in liquid or gas phase using a differentially pumped energy analyzer, major and substantial modifications to the high-vacuum XPS instrumentation are required. In this protocol we describe a membrane-separated cell-based XPS approach that allows characterization of the surface of catalyst nanoparticles dispersed in a flowing liquid or gas (at 2 bar) without any instrumental modification to a high-vacuum X-ray photoelectron spectrometer. The cell features a double-layer graphene membrane that separates a catalyst and its reaction environment from the high-vacuum environment of the high-vacuum XPS system. The graphene membrane is assembled onto the pore of a modified Si₃N₄ window of the cell, admitting an X-ray beam to excite subshell electrons of the catalyst surface atoms in liquid or gas and allowing excited electrons to transit to the high-vacuum environment for XPS analysis. This protocol describes how to create a pore in a Si₃N₄ window, prepare and load graphene layers to seal the pore, assemble the sealed window onto a cell cap, introduce catalyst nanoparticles to the cell cap, install the cell cap to a cell body to form a complete cell, assemble the complete cell to the high-vacuum XPS system, flow liquid or gas through the cell and collect photoelectrons during catalysis or in vivo/in vitro biological processes performed at solid–liquid or solid–gas interfaces in the cell. Equipment and parts setup takes 2–5 d and data collection takes 12–24 h. This protocol examples the operando studies of C–C coupling on Ag nanoparticles performed in flowing liquid and CO oxidation on Ni/TiO₂ nanoparticles in flowing mixture of 0.4 bar CO and 1.6 bar O₂.</p>