The surface of an organism is a dynamic interface that continually adapts to its environment. In nematodes, the cuticle forms a complex boundary that protects against the physicochemical pressures. However, the precise molecular composition and function of this surface remain largely unexplored. By utilizing 3D-OrbiSIMS, an advanced surface-sensitive mass spectrometry method, we directly characterized the molecular composition of the outermost regions (∼50 nm) of