Traceable characterization methods allow for the accurate correlation of the functionality or toxicity of nanomaterials with their underlaying chemical, structural or physical material properties. These correlations are required for the directed development of nanomaterials to reach target functionalities such as conversion efficiencies or selective sensitivities. The reliable characterization of nanomaterials requires techniques that often need to be adapted to the nano-scaled dimensions of the samples with respect to both the spatial dimensions of the probe and the instrumental or experimental discrimination capability. The traceability of analytical methods revealing information on chemical material properties relies on reference materials or qualified calibration samples, the spatial elemental distributions of which must be very similar to the nanomaterial of interest. At the nanoscale, however, only few well-known reference materials exist. An alternate route to establish the required traceability lays in the physical calibration of the analytical instrument’s response behavior and efficiency in conjunction with a good knowledge of the various interaction probabilities. For the elemental analysis, speciation, and coordination of nanomaterials, such a physical traceability can be achieved with X-ray spectrometry. This requires the radiometric calibration of energy- and wavelength-dispersive X-ray spectrometers, as well as the reliable determination of atomic X-ray fundamental parameters using such instrumentation. In different operational configurations, the information depths, discrimination capability, and sensitivity of X-ray spectrometry can be considerably modified while preserving its traceability, allowing for the characterization of surface contamination as well as interfacial thin layer and nanoparticle chemical compositions. Furthermore, time-resolved and hybrid approaches provide access to analytical information under operando conditions or reveal dimensional information, such as elemental or species depth profiles of nanomaterials. The aim of this review is to demonstrate the absolute quantification capabilities of SI-traceable X-ray spectrometry based upon calibrated instrumentation and knowledge about X-ray interaction probabilities.