The effects of anti-solvent addition rate and location, and agitation speed on the metastable zone width of an anti-solvent system were investigated using focused beam reflectance measurement (FBRM) and attenuated total reflectance-Fourier transform infra-red spectroscopy. Benzoic acid in ethanol-water mixtures, with water acting as anti-solvent, was chosen as the model system and was studied at a 500 mL scale. FBRM proved to be the more sensitive method for the detection of nucleation onset. In general, the metastable zone widened with increasing addition rate, with the effect most pronounced when the anti-solvent was added close to the impeller. At this location, an increase in agitation intensity resulted in a narrower metastable zone for all addition rates. For an addition location close to the vessel wall, the metastable zone was narrower and the impact of addition rate and agitation were less pronounced. Substantial variation in the measured metastable zone width was also observed, with nucleation occasionally occurring at bulk concentrations less than the saturation level. It is proposed that the metastable zone width is influenced by the differing degrees of anti-solvent incorporation at each addition location. Close to the impeller anti-solvent is rapidly incorporated leading to consistent results, but, close to the vessel wall, incorporation is hindered by unfavourable mixing conditions leading to premature nucleation and more variability. Computational Fluid Dynamics simulations support this observation. Using the measured metastable zone widths, nucleation kinetics at two different agitation intensities were estimated. Using this data, an agitation dependent expression for the nucleation rate was generated.