Urinary tract infections (UTIs) remain one of the most common bacterial infections worldwide, where timely identification of causative pathogens and their antibiotic susceptibility is critical for guiding therapy. Conventional culture-based methods are reliable but slow, creating an urgent need for rapid, point-of-care (POC) diagnostic solutions. This study aimed to develop and validate a novel Alamar Blue-based system capable of simultaneous Gram classification and antibiotic susceptibility testing (AST) directly from urine samples. The system utilizes Alamar Blue (resazurin), a redox indicator that shifts from violet (oxidized) to pink (reduced) in response to bacterial metabolic activity. Gram differentiation was achieved using two selective antibiotic marker vials: vancomycin/linezolid for Gram-positive and aztreonam/polymyxin B for Gram-negative bacteria. New Gram differentiation standardization (GDS) was performed using the CLSI MIC breakpoint method, ensuring clinical relevance. To improve accuracy, urine artifacts were removed through a filtration approach prior to analysis. Validation on ATCC reference strains demonstrated 100% agreement in Gram classification and 97% similarity with the conventional Kirby–Bauer disc diffusion method. On real urine samples (n = 25), the newly developed GDS predicted 24% of samples as negative, showing 100% concordance with culture-based findings. Furthermore, 16% of samples were identified as true positives, also showing 100% agreement with conventional laboratory reports. AST outcomes produced clear colorimetric readouts within five hours without requiring complex infrastructure. This Alamar Blue-based POC platform provides a rapid, user-friendly, and cost-effective alternative to conventional diagnostics, enabling both Gram differentiation and AST from urine specimens in resource-limited healthcare settings.