In recent years, the nonlinear properties of materials have attracted much interest in nondestructive testing and in ultrasound diagnostic applications. Acoustic nonlinear parameters represent an opportunity to improve the information that can be extracted from a medium such as structural organization and pathologic status of tissue. In this paper, a method called pulse subtraction intermodulation (PSI), based on a multipulse technique, is presented and investigated both theoretically and experimentally. This method allows separation of the intermodulation products, which arise when 2 separate frequencies are transmitted in a nonlinear medium, from fundamental and second harmonic components, making them available for improved imaging techniques or signal processing algorithms devoted to tissue characterization. The theory of intermodulation product generation was developed according the Khokhlov-Zabolotskaya-Kuznetsov (KZK) nonlinear propagation equation, which is consistent with experimental results. The description of the proposed method, characterization of the intermodulation spectral contents, and quantitative results coming from in vitro experimentation are reported and discussed in this paper.