Human rhinoviruses are responsible for the majority of virus-induced asthma exacerbations, contributing to substantial health problems in adults and children and leading to a significant number of deaths each year. Upon infection, the human rhinovirus encoded proteases 2Apro and 3Cpro cause extensive damage to the host cell, including disruption of nucleocytoplasmic transport and shutting down host transcription and translation. Despite current knowledge regarding the activity of both 2A and 3C proteases, a complete picture of the events that occur during infection has not been elucidated; this is in part due to a lack of reliable antibodies available for detection of 2Apro in particular. In order to enable identification of 2Apro during infection and therefore aid investigations of 2Apro activity, we have generated a recombinant HRV16 genome, which enables expression of a HA tag at the C-terminus of 2Apro. The recombinant virus can be produced at titres comparable to wildtype HRV16, is able to infect host cells with the same kinetics as wildtype HRV16 and the HA-tagged 2Apro can be detected by Western blot and immunofluorescence. We have subsequently used this recombinant virus to examine the kinetics of 2Apro activity during infection with HRV16.