OBJECTIVE The aim of this paper was to quantify the analysis error introduced by iterative closest point (ICP) image registration. We also investigated whether a subsequent subtraction process can reduce process error. METHODS We tested metrology and two 3D inspection software using calibration standards at 0.39 μm, and 2.64 μm and mathematically perfect defects (softgauges) at 2 and 20 μm, on free form surfaces of increasing complexity and area, both with and without registration. Errors were calculated in percentage relative to the size of the defect being measured. Data were analysed in GraphPad Prism 9, normal and two-way ANOVA with post-hoc Tukey's was applied. Significance was inferred at p < 0.05. RESULTS Using ICP registration introduced errors from 0 % to 15.63 % of the defect size depending on the surface complexity and size of the defect. Significant differences were observed in analysis measurements between metrology and 3D inspection software and within different 3D inspection software, however, one did not show clear superiority over another. Even in the absence of registration, defects at 0.39 μm, and 2.64 μm produced substantial measurement error (13.39-77.50 % of defect size) when using 3D inspection software. Adding an additional data subtraction process reduced registration error to negligible levels (<1 % independent of surface complexity or area). CONCLUSIONS Commercial 3D inspection software introduces error during direct measurements below 3 μm. When using an ICP registration, errors over 15 % of the defect size can be introduced regardless of the accuracy of adjacent registration surfaces. Analysis output between software are not consistently repeatable or comparable and do not utilise ISO standards. Subtracting the datasets and analysing the residual difference reduced error to negligible levels. CONCLUSIONS This paper quantifies the significant errors and inconsistencies introduced during the registration process even when 3D datasets are true and precise. This may impact on research diagnostics and clinical performance. An additional data processing step of scan subtraction can reduce this error but increases computational complexity.