OBJECTIVE Up to 85% of women with interstitial cystitis/bladder pain syndrome have pelvic floor dysfunction and hypertonicity. Current evaluation methodologies lack objective measures of pelvic floor muscle activity. We examined the ability of using intravaginal high-density surface electromyography to quantitatively, objectively and noninvasively map pelvic floor muscle activity and innervation zone locations in patients with interstitial cystitis/bladder pain syndrome. METHODS Fifteen women with interstitial cystitis/bladder pain syndrome and 15 controls underwent 2 sessions of digital pelvic examinations and high-density surface electromyography assessments. The root mean squared amplitude of high-density surface electromyography was first calculated, and the resting root mean squared ratio was then calculated by normalizing the resting electromyography root mean squared to the peak electromyography amplitude reached during maximum voluntary contraction. Innervation zone distributions were obtained from decomposed high-density surface electromyography signals. The correlation between the root mean squared ratio and interstitial cystitis/bladder pain syndrome symptom scores and pelvic floor muscle alignment were investigated in patients with interstitial cystitis/bladder pain syndrome and healthy controls. RESULTS Women with interstitial cystitis/bladder pain syndrome demonstrated significantly increased resting root mean squared ratios compared to controls (0.155±0.048 vs 0.099±0.041, p=0.0019). Significant correlations were found between resting root mean squared ratio and patient reported pain (rs=0.523, p=0.003), interstitial cystitis symptom (rs=0.521, p=0.003) and problem indices (rs=0.60, p <0.001). In addition, women with interstitial cystitis/bladder pain syndrome were more likely to have shortened pelvic floor muscles (80%, 12 vs 13.3%, 2, p <0.01). Women with shortened pelvic floor muscles demonstrated significantly higher resting root mean squared ratio compared to those with normal pelvic floor muscle length (0.155±0.046 vs 0.107±0.040, p=0.0058). CONCLUSIONS Intravaginal high-density surface electromyography offers an objective and quantitative strategy to noninvasively assess pelvic floor muscle dysfunction in women with interstitial cystitis/bladder pain syndrome. Abundant spatiotemporal muscle activity information captured by high-density surface electromyography allows for mapping innervation zone distributions for major pelvic floor muscles.