Background/Objectives: Vancomycin hydrochloride (HCl) for injection is a vital antibiotic for severe infections. Vancomycin is an indispensable first-line antibiotic for life-threatening infections like methicillin-resistant Staphylococcus aureus (MRSA) bacteremia and serves as a key oral agent for severe Clostridium difficile infection. This makes rigorous quality monitoring essential. Concerns about the efficacy of certain generic vancomycin HCl products have prompted regulatory agencies to increase post-market evaluations to ensure patient safety. Aligned with these efforts, this study aimed to comparatively evaluate the potency and quality of five randomly procured generic intravenous vancomycin HCl products marketed in Saudi Arabia. Methods: Using both microbiological and chemical methodologies as recommended by the US Pharmacopeia (USP) guidelines. Results: The microbiological assay, utilizing the USP “Antibiotics—Microbial Assay” method using Bacillus subtilis ATCC 6633 via the agar diffusion technique, demonstrated a strong linear correlation (R² > 0.98) between inhibition zone diameters and vancomycin concentration. All five tested generics products showed compliance (~ 105%) with USP biological activity standards. For chemical quantification, a High-Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) method was developed and validated according to ICH Q2(R2) guidance. This method showed excellent linearity (R² = 0.998), accuracy (96.9–102%), and good precision. Conclusions: Both methods confirmed the potency of the tested generics within USP limits. However, minor variations observed between microbiological and chemical results highlight the importance of employing complementary techniques for comprehensive quality assessment. These findings underscore the importance of robust regulatory frameworks and ongoing post-market surveillance to ensure the continued safety, efficacy, and availability of quality-assured generic antibiotics in the Saudi market.