The laser ranging interferometer (LRI) on board of the GRACE follow-on spacecraft, launched in May 2018, is the first laser interferometer to perform an inter-satellite range measurement. It is designed for ranging noise levels of 80 nm Hz−1/2 for frequencies above 20 mHz, i.e., about a ten-fold improvement with respect to the GRACE follow-on main microwave ranging instrument. One of the most critical steps during the commissioning phase of the instrument is the so-called initial line of sight calibration procedure (or initial acquisition). This process is required to quantify large uncertainties with respect to laser beam pointing angles and laser frequency, which must be known to establish the interferometer link. It is a nine hour scan of five degrees of freedom, which all need to match simultaneously at least once. Here we report on laboratory tests to further validate the calibration procedure using a mock-up LRI and a set-up, the so-called laser link simulator, that creates conditions similar to those with ~220 km distance between the SC. The experiments presented here made use of LRI-like hardware and software and were carried out recreating critical conditions such as received laser powers on the pico-Watt level and their dependence on the SC misalignments, flat-top beams as receiving beams and Doppler frequency shifts. Several configurations were tested, including a full line of sight calibration with angular scans in both mock-up SC and frequency scan in one of the lasers. Results are well in agreement with the expectations and confirm, well before the LRI commissioning phase, the robustness of the procedure under realistic conditions, which had not yet been fully tested experimentally.