The multidrug resistance (MDR) phenotype is frequently associated with the overexpression of transmembrane drug proteins such as the P-glycoprotein (Pgp) and/or multidrug resistance related protein-1 (MRP1). These proteins belong to the superfamily of the so-called ATP-binding cassette superfamily and act as drug efflux pumps of a broad range of chemotherapeutic agents commonly used in the treatment of malignancies. These proteins have been found to be overexpressed in both haematological and solid tumours and are considered as adverse prognostic factors. The ability to obtain in vivo and non-invasively information regarding the functional activity of MDR-related transporters, using probes that mimic the antineoplastic agents, provide a very useful tool in the clinical setting by determining the individual tumour susceptibility to chemotherapy. This previous knowledge could serve as a critical tool for optimizing chemotherapeutic protocols on a patient-specific basis. The emergence of non-invasive molecular imaging techniques using radiolabelled probes provides an interesting approach for functional assessment of the classical mechanism of MDR in cancer patients. Toward this objective, the clinically approved 99mTc-labelled cationic lipophilic complexes (sestamibi and tetrofosmin) have been characterized as transport substrates of Pgp and MRP1 and proposed as surrogate markers of chemotherapeutic agents for functional evaluation of MDR by single-photon emission tomography (SPECT). Here we review the potential applications of these agents in identifying drug resistance mechanisms based on functional assays and their potential as a tool for evaluating the efficacy of MDR inhibitors, using cellular and animal models of chemoresistance.