Helicobacter pylori infection remains one of the most prevalent infections worldwide, especially in low-resource countries, and the major risk factor for peptic ulcer and gastric cancer. The “test-and-treat” strategy is recommended by several guidelines and consensus. The choice of testing method is based on patient age, presence of alarm signs and/or symptoms, use of non-steroidal anti-inflammatory drugs, as well as local availability, test reliability, and cost. Culture is the gold standard to detect H. pylori and, possibly, to perform susceptibility testing, however, it requires upper endoscopy and dedicated labs. Recent advances in molecular biology have provided new strategies in detecting infection and antimicrobial resistance without invasive tests. In this review we attempt to offer a comprehensive panorama on the new diagnostic tools and their potential use in clinical settings, in order to accomplish specific recommendations.