Exercise increases the synthesis of collagen in the extracellular matrix of skeletal muscle. Breakdown of skeletal muscle collagen has not yet been determined because of technical limitations. The purpose of the present study was to use local sampling to determine skeletal muscle collagen breakdown. Microdialysis fibers were tested in vitro to predict bath hydroxyproline (OHP) concentrations. We used an N-methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide derivative to analyze OHP using gas chromatography–mass spectroscopy (GC–MS) and compared the results with a colorimetric OHP assay. Ten young, healthy male subjects performed a bout of resistance exercise with one leg, followed 17–21 h later by in vivo skeletal muscle sampling by microdialysis in exercised (EX) and control (CON) legs. Microdialysis reliably predicted [OHP] in vitro (R2=0.90). Analysis with GC–MS was strongly correlated to traditional analysis methods (CON: slope=1.03, R2=0.896, and P<0.05, EX: slope=0.795, R2=0.896, and P<0.05). We conclude that in vitro, microdialysis fibers were able to measure OHP concentrations and were sensitive to changes in concentrations, a strenuous bout of exercise did not increase skeletal muscle collagen breakdown 17–21 h post-exercise, and our measurement of OHP using GC–MS was in agreement with traditional assays.