Introduction: Gut microbiome and microbiota play a vital role in human health and influence various physiological processes. Alterations in the composition of the gut microbiota are associated with various diseases. Understanding the gut microbiome is crucial for personalized nutrition and health care. This study reviewed various techniques for detecting the gut microbiome and evaluated the sensitivity and specificity of selected tests used in a tertiary care hospital in Pakistan. Methodology: Data from patients with gastrointestinal issues were retrospectively analyzed considering demographic information, symptoms, and test results. The sensitivity and specificity were determined for three techniques: Shotgun Metagenomics, Breath Tests, and Single-Cell Sequencing. Results: The study included 57.3% males, with most patients aged 40–50 years. Shotgun Metagenomics demonstrated the highest sensitivity (0.936) and specificity (0.667), followed by Single-Cell Sequencing with sensitivity (0.901) and specificity (0.762), whereas Breath Tests had lower sensitivity (0.794) and specificity (0.483). The area under the ROC curve was the highest for Shotgun Metagenomics (0.675), indicating its potential as a diagnostic tool for gut microbes. Conclusion: Understanding the impact of the gut microbiome on human health is a rapidly evolving field, with promising diagnostic techniques, such as Shotgun Metagenomics, demonstrating high sensitivity and specificity. Future studies should continue to explore and refine these methods to improve healthcare and personalize nutritional interventions.