For a long time, the diagnostic approach to IA/Aspergillus disease has been a challenge. The definitive diagnosis is made by correct microbiological and/or histopathological documentation, undoubtedly the fundamental cornerstone for therapeutic decision-making. However, it is considered limited by lack of sensitivity and speed, often being counterproductive, as in many cases invasive procedures are needed (e.g., fibrobronchoscopy [FBC] or tissue biopsy), which delays treatment and undermines survival of at-risk patients. This highlights the need for faster and more accurate diagnostic tools. Although novel serological and molecular methods have been developed that have demonstrated their potential to replace conventional diagnostic tests, inconsistencies in interpretation and validation among the different approaches limit their reproducibility and large-scale clinical application. However, whenever possible, an immunological and/or molecular diagnosis should be made, as it has proven useful in different detection and diagnostic strategies, which with an understanding of its strengths and limitations, and the standardization of the different techniques available, can be incorporated into care protocols and diagnostic algorithms, as an aid in administering and monitoring the different antifungal treatments and predicting possible clinical outcomes. Due to the limited sensitivity (SE) of many of the tests, and variations in the specificity (SP) of some of them, the combined use of several diagnostic tools during the high-risk period for invasive infection and/or during the early period in the course of IA/Aspergillus disease would allow an adequate diagnostic approach, provided that their results are interpreted together with the existing clinical and imaging information.