Non-culture-based diagnostics have been developed to help establish an early diagnosis of invasive fungal infection. Studies have shown that these tests can significantly impact the diagnosis of infection in high risk patients. Aspergillus galactomannan EIA testing is well-recognized as an important adjunct to the diagnosis of invasive aspergillosis and can be detected in serum, bronchoalveolar lavage and other fluids. Galactomannan testing used along with PCR testing has been shown to be effective when integrated into care paths for high risk patients for both diagnoses and as a surrogate marker for outcome when used in serial testing. Beta-d-glucan assays are non-specific for several fungal genera including Aspergillus and Candida and in high risk patients have been an important tool to augment the diagnosis. Lateral flow technology using monoclonal antibodies to Aspergillus are available that allow rapid testing of clinical samples. While standard PCR for Candida remains investigational, T2 magnetic resonance allows for the rapid diagnosis of Candida species from blood cultures. Aspergillus PCR has been extensively validated with standardized approaches established for these methods and will be included in the diagnostic criteria in the revised European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC-MSG) definitions. Finally, these non-culture-based tests can be used in combination to significantly increase the detection of invasive mycoses with the ultimate aim of establishing an early diagnosis of infection.