Nanofocus X-ray projection imaging technology with a resolution of 0.25 μm has been developed and applied to the estimation of the profile of through-silicon vias (TSVs) several microns in diameter. However, analysis and examination of the uncertainty of the system and the calibration method for measurement have not been properly discussed thus far. These topics should be discussed in consideration of the actual application of the method to the automation of inline inspection and the measurement processes of TSV devices. This study focuses on the quantitative analysis of the uncertainty budget in the measurement of the whole X-ray microscope system. A calibration method using a known, conventionally defined TSV sample as a calibration device is employed. The uncertainties are divided into calibration, mechanical, electrical, and algorithmic factors, and their contributions to the combined standard uncertainty and the expanded uncertainty are estimated. An actual case for the analysis of the uncertainty budget is evaluated, where the profile is estimated for actual images with a signal-to-noise ratio of 2.2.