Introduction: Urethral pain syndrome (UPS) is still a pathology in which the diagnosis is formulated as a “diagnosis of exclusion”. The exact pathogenetic mechanisms are not yet fully understood and clear recommendations for the prevention and treatment of UPS are absent. Methods and Participants: A clinical and laboratory evaluation of 55 patients with established UPS included history taking, basic laboratory tests (e.g., complete blood count and clinical urine test), physical examination, uroflowmetry, and cystourethroscopy. Additionally, transvaginal ultrasound (TVUS) with compression elastography and cross-polarization optical tomography (CP OCT) were performed in 24 and 33 patients with UPS, respectively. The control group consisted of 14 patients with no complaints from the urinary system. Results: TVUS showed an expansion in the diameter of the internal lumen of the urethra, especially in the proximal region compared with the norm. Compression elastography revealed areas with increased stiffness (presence of fibrosis) in urethral and surrounding tissues. The performed CP OCT study showed that in UPS, the structure of the tissues in most cases was changed: trophic alterations in the epithelium (hypertrophy or atrophy) and fibrosis of underlying connective tissue were observed. The proximal fragment of the urethra with UPS underwent changes identical to those of the bladder neck. Conclusion: This paper showed that the introduction of new technology—CP OCT—in conjunction with TVUS will allow verification of structural changes in tissues of the lower urinary tract at the level of their architectonics and will help doctors understand better the basics of the UPS pathogenesis.