A V-shape Bio-Transfer-Standard (V-BTS), developed and produced at the University of Helsinki (UH), was measured in two laboratories. In comparison to Siemens Star calibration specimens, the V-BTS performs better at high lateral frequencies close to the diffraction limit of the optical instrument. This permits determining of the Instrument Transfer Function (ITF). The V-BTS features two lipid bilayer steps that partly overlap each other at an angle of 20°, with an average height of 4.6 ± 0.1 nm. The Round Robin (RR) test aims to determine whether the V-BTS and the developed application protocol work with different optical profilers in different laboratories. First the artefact was measured at Sensofar-Tech, S.L. using an S-neox profiler working in Phase Shifting Interferometry mode. Then V-BTS was measured at UH using a custom-built Scanning White Light Interferometer. All measurements done by four different operators at the two laboratories have a range or standard deviation of ±0.1 nm which agrees with the theoretical estimates and with measurements done using an atomic force microscope and with a surface plasmon resonance based instrument. The RR results show the applicability of the V-BTS for calibration and for ITF characterization of 3D optical profilers.