Ultrasonography (US) of the nail is raising interest in the last years and its feasibility, quickness and amount of descriptive data may provide valuable information. Different authors presented several scanning approaches to nail complex in different pathological conditions, such as psoriasis, but no scanning protocol was ever proposed using healthy subjects as population of reference. The aim of the study was to establish a protocol for the US of nail plate and to assess whether the measurement of the nail plate is influenced by longitudinal vs transverse scan, sex, digit and hand dominance. Using high frequency probe and a Canon Aplio i800 machine, ultrasonographers took scans of nail plates of the hands from healthy subjects. Nail plate shape, thickness and power Doppler signal (PDUS) were evaluated and scans were taken both on longitudinal and transverse axis, at distal, middle and proximal portion of the nail plate or at a fixed angles of − 45°, 0° or + 45°. All the images were then revised and scored using a DICOM software, in order to allow good standards of accuracy and reproducibility. A total of 27 subjects (14 females and 13 males) were assessed. The measures did not result to differ in different portions or angles. Furthermore, no difference appears in sex or dominant vs not dominant hand. A decreasing and significant trend for nail plate thickness was found from the first to the fifth finger. Doppler signal was found in all but one subjects, with a range from almost absent to very evident. No difference was found between groups regarding PDUS. The data provided suggest that a proper scan protocol should include all the nails and evaluation should be done both on longitudinal and transverse axis. Since Doppler signal is highly variable in healthy subjects, its presence should be carefully considered as pathological finding. Observations provided by this study clarify important points of the scanning technique and solve doubts related to which nails should be scanned and where to evaluate quantitative parameters.