Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major global health threat, compounded by the rise of extensively drug-resistant (XDR) and multidrug-resistant (MDR) strains. This review critically examines the current landscape of laboratory diagnostic methods for MTB, encompassing both established techniques and recent advancements. We explore the growth and genetic characteristics of MTB that underpin drug resistance development and detection. We then provide a comparative analysis of smear microscopy, culture-based methods, antigen detection, molecular diagnostics (including nucleic acid amplification tests and whole-genome sequencing), spectroscopic techniques (such as Raman spectroscopy), and mass spectrometry-based approaches. Notably, this review focuses on pathogen-based diagnostic methods, excluding host immune response assays. The strengths and limitations of each method are evaluated in terms of sensitivity, specificity, turnaround time, cost-effectiveness, and suitability for resource-limited settings. Finally, we discuss the future of TB diagnostics, emphasizing the need for integrated, multi-modal platforms, the incorporation of artificial intelligence (AI) for enhanced data analysis, and the development of affordable, point-of-care testing to improve accessibility and impact in high-burden regions. Overcoming current diagnostic challenges is essential for improving patient outcomes and achieving global TB elimination goals.