Mass gauging of fluids in space systems (such as propellant tanks in upper stages and satellites, or life support systems) is a challenging task due to the absence of buoyancy. The vast number of possible liquid–gas configurations makes some current mass gauging ground techniques unreliable. The Spectral Mass Gauging technique has been recently presented as a promising approach for mass gauging in microgravity conditions given its independence on the liquid configuration. In this technique, the volume of liquid in a tank is determined from the spectral analysis of the frequency response of the tank walls to an acoustic excitation. We present an experimental study of the technique in terrestrial gravity and microgravity. Experiments in weightlessness were carried out in 15 drops at the drop tower of the Center of Applied Space Technology and Microgravity (ZARM) in Germany. We provide a criterion for the spectral analysis based on some characteristics of the Fast Fourier Transform of the acquired data, namely the Root Mean Square and the prominence of the spectral peaks. This criterion is used to study the effects of the acoustic actuation characteristics and the location of the data acquisition devices on the application of the acoustic technique in the considered scenarios. Our analysis also shows an independence of the technique on the gravity level, which confirms the feasibility of its use in space. The influence of the tank size on the applicability of this technique is discussed too.