Differential Acoustic Resonance Spectroscopy (DARS) has been developed to investigate the acoustic properties of samples in the kilohertz frequency range. This new laboratory measurement technique examines the change in resonant frequencies of a cavity perturbed by the introduction of a small test sample. The resonant frequency shift between the empty and sample‐loaded cavity is used to estimate the acoustic properties of the loaded sample. This paper presents a DARS perturbation formula that combines a theoretical derivation with numerical simulation and laboratory measurements. Furthermore, a semi‐empirical calibration technique is proposed to estimate the acoustic properties of a test sample. This research demonstrates the potential of the DARS measurement technique for estimating the acoustic properties of acoustically small and/or irregularly shaped samples.