The economic viability of low temperature fuel cells as clean energy devices is enhanced by the development of inexpensive oxygen reduction reaction catalysts. Heat treated iron and nitrogen containing carbon based materials (Fe–N/C) have shown potential to replace expensive precious metals. Although significant improvements have recently been made, their activity and durability is still unsatisfactory. The further development and a rational design of these materials has stalled due to the lack of an in situ methodology to easily probe and quantify the active site. Here we demonstrate a protocol that allows the quantification of active centres, which operate under acidic conditions, by means of nitrite adsorption followed by reductive stripping, and show direct correlation to the catalytic activity. The method is demonstrated for two differently prepared materials. This approach may allow researchers to easily assess the active site density and turnover frequency of Fe–N/C catalysts.