Effective management of bacterial infections relies on timely diagnosis and appropriate antibacterial therapy. In vitro antimicrobial susceptibility testing (AST) is a cornerstone for guiding such treatments, enabling the determination of bacterial resistance and informing clinical decisions. Classical AST methods, such as agar or broth dilution and disk diffusion, remain gold standards due to their simplicity, cost-effectiveness, and reliability. However, their limitations, including time-intensive protocols and limited insights into antimicrobial resistance (AMR) mechanisms, underscore the need for advanced methodologies. Emerging techniques, such as microfluidics-based platforms, offer rapid, high-throughput, and precise minimum inhibitory concentration (MIC) determinations. These systems can replicate complex experimental conditions while analyzing single-cell resistance mechanisms, making them promising for both research and clinical diagnostics. Osteoblast infection models, which simulate in vivo like conditions, provide unique insights into bacterial–host interactions and AMR in tissue-level infections, especially those associated with implant-related complications. Similarly, biofilm models are indispensable for understanding resistance patterns in bacterial biofilms, which differ significantly from planktonic states. Incorporating biomaterials such as bone graft substitutes into biofilm models can improve their relevance to real-world infections and aid in the evaluation of innovative antimicrobial strategies. Despite their potential, advanced models face challenges such as high costs, technical complexity, and limited validation for diverse bacterial strains. Future developments must focus on integrating these innovations with traditional methods to enhance adaptability across research and clinical applications. Addressing infections involving intracellular bacteria, recurrent biofilm formation, and novel AMR mechanisms requires continuous refinement of AST methodologies. This chapter comprehensively reviews the evolution, applications, and challenges of AST, highlighting its critical role in combating the global threat of AMR. Emphasis is placed on the need for robust, versatile, and clinically relevant AST systems to improve therapeutic strategies and patient outcomes.