Ventilator-associated pneumonia (VAP) is a serious infectious condition in intensive care unit (ICU) patients, currently related to a high mortality rate. Therefore, this complication of mechanical ventilation requires a prompt diagnosis and adequate antibiotic treatment. The detection of the causative organism is imperative for guiding an appropriate therapy as there is strong evidence of the adverse effect of inadequate empirical treatment on outcome. The major difficulty of the microbial investigation is to obtain the sample from the lower respiratory tract, mainly because of the potential contamination with upper airways flora, which may result in a misinterpretation of the cultures.Microbial investigation in VAP is based on the culture of samples obtained from lower respiratory tract by noninvasive or invasive methods. The most common techniques of sampling are the endotracheal aspirate (ETA), which is considered a noninvasive method, the protected specimen brush (PSB) and the bronchoalveolar lavage (BAL), both being invasive methods of investigation. The latter were designed as an attempt to avoid the colonizing flora of the upper airways. The best of these diagnostic approaches is still controversial. In terms of outcome, there is strong evidence that the impact of both invasive and noninvasive methods seems to be similar. In terms of cost, however, the endotracheal aspirate is less expensive compared to BAL or PSB. On the other hand, invasive methods could be particularly beneficial in patients who are not responding to the initial empirical antibiotic treatment.The rationale for the quantitative culture of the respiratory samples is to differentiate between infection and colonization of lower airways, because the bacterial colonization is a frequent event in mechanically ventilated patients. The thresholds currently employed for the diagnosis of the pneumonia are the following: ETA samples, ≥105–106colony forming units (cfu)·mL−1; PSB samples, ≥103cfu·mL−1; and BAL samples, ≥104 cfu·mL−1.Intending to provide a practical approach to the issue, the present manuscript reviews the available noninvasive (blood culture, endotracheal aspirate) and invasive (protected specimen brush, bronchoalveolar lavage, blinded methods and lung biopsy) techniques used for the diagnosis of ventilator-associated pneumonia.