Background: Functional near infrared spectroscopy (fNIRS) is a versatile, noninvasive, and inexpensive tool that can be used to measure oxyhemoglobin (O2Hb) changes in the cortical brain caused by increasing bladder sensation during filling in upright posture. This study’s purpose is to provide a rigorous methodologic template that can be implemented for comparative studies of fNIRS in the diagnosis and management of lower urinary tract symptoms including overactive bladder (OAB) and other forms of lower urinary tract dysfunction. Methods: Participants without any urologic conditions completed a validated oral hydration protocol facilitating and equilibrating natural bladder filling. First desire to void and real time bladder sensation (0–100%) were recorded using a Sensation Meter. A 24-channel fNIRS template simultaneously recorded prefrontal cortical O2Hb. Each channel was analyzed between “first desire” to void and 100% sensation, defined in this study as the period of “high sensation”. Channels were sub-divided by cortical regions: right (nine channels), left (nine channels), middle (six channels). Results: A total of eight participants (male: n=4, female: n=4) were enrolled with mean age 39±19.9 years and body mass index (BMI) of 25±3.93 kg/m2. There were no differences in age, BMI, race, or OAB survey scores based on biological sex. Signal acquisition improved with power bank use, postural head support for motion reduction, and head cap optimization. Acceleration-based concurrent motion measurement was effectively utilized to remove motion artifacts. O2Hb concentration patterns appeared irregular during low sensation and increased during high sensation after first desire across the frontal cortex. Conclusions: Employing a stepwise approach, this study defined a methodological guide for improved prefrontal fNIRS signal acquisition and analysis during bladder filling. The technique demonstrated that prefrontal fNIRS cortical O2Hb increases with elevated bladder sensation in normal subjects and sets the stage for comparative studies in individuals with OAB and other forms of lower urinary tract dysfunction.