Bladder cancer (BCA) is relatively common and potentially recurrent/progressive disease. It is also costly to detect, treat, and control. Definitive diagnosis is made by examination of urine sediment, imaging, direct visualization (cystoscopy), and invasive biopsy of suspect bladder lesions. There are currently no widely-used BCA-specific biomarker urine screening tests for early BCA or for following patients during/after therapy. Urine metabolomic screening for biomarkers is costly and generally unavailable for clinical use. In response, we developed Raman spectroscopy-based chemometric urinalysis (Rametrix™) as a direct liquid urine screening method for detecting complex molecular signatures in urine associated with BCA and other genitourinary tract pathologies. In particular, the RametrixTM screen used principal components (PCs) of urine Raman spectra to build discriminant analysis models that indicate the presence/absence of disease. The number of PCs included was varied, and all models were cross-validated by leave-one-out analysis. In Study 1 reported here, we tested the Rametrix™ screen using urine specimens from 56 consented patients from a urology clinic. This proof-of-concept study contained 17 urine specimens with active BCA (BCA-positive), 32 urine specimens from patients with other genitourinary tract pathologies, seven specimens from healthy patients, and the urinalysis control SurineTM. Using a model built with 22 PCs, BCA was detected with 80.4% accuracy, 82.4% sensitivity, 79.5% specificity, 63.6% positive predictive value (PPV), and 91.2% negative predictive value (NPV). Based on the number of PCs included, we found the RametrixTM screen could be fine-tuned for either high sensitivity or specificity. In other studies reported here, RametrixTM was also able to differentiate between urine specimens from patients with BCA and other genitourinary pathologies and those obtained from patients with end-stage kidney disease (ESKD). While larger studies are needed to improve RametrixTM models and demonstrate clinical relevance, this study demonstrates the ability of the RametrixTM screen to differentiate urine of BCA-positive patients. Molecular signature variances in the urine metabolome of BCA patients included changes in: phosphatidylinositol, nucleic acids, protein (particularly collagen), aromatic amino acids, and carotenoids.