During the long shutdown 2 of the LHC, the LHCb Experiment was upgraded to a trigger-less system reading out the full detector at 40 MHz bunch crossing rate with all selection algorithms executed in a CPU farm. The upgraded Vertex Locator (VELO) of the LHCb is a hybrid pixel detector read out by the “VeloPix” front-end ASIC with on-chip zero-suppression. This paper describes a novel way of calibrating the VELO detector based on dedicated threshold scan firmware. The firmware is implemented in the control and data acquisition back-end boards of the detector. This threshold scan method has multiple advantages compared with the default one: it is faster as the algorithm was moved from software to firmware, it is more deterministic by minimizing software interruptions and data transfer and it is more precise by reducing noise due to ASIC operation.