Polymerase Chain Reaction is one of the most common molecular methods used in disease diagnosis. It ensures rapid, specific, and sensitive pathogen detection. The current chapter discusses the problems encountered mainly when PCR amplification is required for diagnostic applications. It covers the existing limitations and practical solutions and touches upon problematic issues, such as how PCR can be a transformative tool in detecting various diseases and their causes. The PCR reaction is detailed, showing the core components and mechanisms. Summarizing several types of PCR, their descriptions, and applications provides a comprehensive perspective on this versatile technique. Since the capabilities of PCR are versatile, there are some inherent limitations involving susceptibility to contamination, inhibition, and the need for high-quality nucleic acids. The amplification factors of PCR include primer design, annealing temperature, and template quality. This also comes out in considerable detail. The chapter includes false positives and negatives and the role of controls and optimization to achieve better quality diagnostics. Some common PCR problems, such as non-specific amplification and low yield, are highlighted, along with strategies for troubleshooting them. Other issues encountered during agarose gel electrophoresis, such as smearing and poor resolution, are discussed, along with practical solutions given for most problems encountered. The chapter ends by acknowledging the critical role of PCR in disease diagnosis and, at the same time, underlines the need to understand the challenges and limitations associated with it. It argues further for continuous progress in PCR technology and for the improvement of rigorous methodological improvement that will give PCR its maximum diagnostic potential and reliability. This elaborate review might serve as a guide for researchers and clinicians in harnessing PCR for disease detection.