The presence of off-flavor compounds in fish represents a significant economic problem encountered in aquaculture production. The off-flavor compounds are due to the absorption of substances produced by a range of microorganisms. Currently, a number of strategies have been used to prevent or limit the growth of these microorganisms. Therefore, it is important to evaluate the effectiveness of strategies via monitoring the concentrations of off-flavor compounds in the recirculating aquaculture system. In vivo solid-phase microextraction (SPME), a rapid and simple sample preparation method, will allow monitoring the concentration of off-flavor compounds in live fish. In this research, geosmin and 2-methylisoborneol (2-MIB) produced by cyanobacteria and actinomycetes, which are the major sources for "earthy" and "muddy" flavors in fish, were selected as representatives. In order to accurately quantify these compounds in fish muscle, two kinetic calibration methods, on-fiber standardization and measurement using predetermined sampling rate, were used as quantification methods, which were both validated by traditional methods. The detection limit of in vivo SPME in fish muscle was 0.12 ng/g for geosmin and 0.21 ng/g for 2-MIB, which are both below the human sensory thresholds.