Flow systems such as a BIAcore biosensor can be very efficient tools to isolate high affinity antibody fragments from affinity matured phage display libraries. Here we show that using flow based selection, we can readily isolate a variant with a 35-fold higher affinity, especially with a 7 fold better off-rate, compared to the parent clone after only a single round of selection from a second generation affinity matured phage display library. The flow system represents a fast method to isolate affinity improved antibody fragments and can be particularly useful for isolating antibodies to antigens that have poor solubility, are toxic to the host cell, or prone to aggregation.