Enzymatic detergents are widely used to clean gastrointestinal endoscopes before high-level disinfection. Although proper use of enzymatic detergents may appear straightforward, common misuses include failure to dilute the enzymatic detergent, overdilution of enzymatic detergent, use of expired enzymatic detergent, inadequate exposure time, and failure to rinse the enzymatic detergent from the instrument being cleaned. Each type of misuse has its own rationale. In these studies, the authors show by high-performance liquid chromatography that the type of enzymatic detergent and the dilution and rinsing of enzymatic detergents affect the amount of residual high-level disinfectant (ortho-phthalaldehyde) left on test segments of flexible endoscope insertion tubes. The authors also qualitatively demonstrate that proteinaceous material, which is stained a dark color by ortho-phthalaldehyde (OPA), remains on colonoscopes that have been cleaned with improperly diluted or rinsed enzymatic detergents. These findings emphasize the importance of diluting and using enzymatic detergents exactly as directed by their manufacturers to reduce bioburden and residual amounts of high-level disinfectant on flexible endoscopes.