Antibiotic contamination of drinking water and sewage is a matter of environmental and public health concern. Traditionally, ELISA or HPLC methods have been used to detect and measure antibiotic contamination. By applying an optical biosensing method, biolayer inteferometry (BLI), we have developed a kinetic competition binding assay capable of quantitating less than lppm (~33 μM) amoxicillin. Similar to surface plasmon resonance, BLI senses changes that occur upon binding of one molecule to another near a surface to measure association and dissociation. Immobilized amoxicillin was used to screen for binding against an analyte solution of anti-amoxicillin equilibrated with amoxicillin-containing water samples, yielding binding that fit a one-state model. Maximal binding correlated highly with amoxicillin concentration. Simplified analysis of samples from water and sewage treatment plants in Georgia allowed quantitation without kinetic modeling. The assay is sensitive, cost-effective, fast and readily adaptable to a variety of samples and other small molecules.