Invasive aspergillosis occurs in a wide range of clinical scenarios, is protean in its manifestations, and is still associated with an unacceptably high mortality rate. Early diagnosis is critical to a favourable outcome, but is difficult to achieve with current methods. Deep tissue diagnostic specimens are often difficult to obtain from critically ill patients. Newer antifungal agents exhibit differential mould activity, thus increasing the importance of establishing a specific diagnosis of invasive aspergillosis. For these reasons, a range of alternate diagnostic strategies have been investigated. Most investigative efforts have focused on molecular and serological diagnostic techniques. The detection of metabolites produced by Aspergillus spp and a range of aspergillus-specific antibodies represent additional, but relatively underused, diagnostic avenues. The detection of galactomannan has been incorporated into diagnostic criteria for invasive aspergillosis, reflecting an increased understanding of the performance, utility, and limitations of this technique. Measurement of (1,3)-beta-D glucan in blood may be useful as a preliminary screening tool for invasive aspergillosis, despite the fact that this antigen can be detected in a number of other fungi. There have been extensive efforts directed toward the detection of Aspergillus spp DNA, but a lack of technical standardisation and relatively poor understanding of DNA release and kinetics continues to hamper the broad applicability of this technique. This review considers the application, utility, and limitations of the currently available and investigational diagnostic modalities for invasive aspergillosis.