Overexpression of ecto-5-nucleotidase is found to be linked to cancer progression and other diseases which makes screening of its inhibitors on demand. Unfortunately, there is scarcity of reported ecto-5-nucleotidase inhibitors because of unavailability of simple, fast and efficient methods to study its inhibition. In this study, we have developed a very fast, reverse polarity capillary electrophoresis based method for screening of ecto-5-nucleotidase inhibitors. Both EOF and electrophoretic mobility were maintained in same direction by dynamically coating lining of capillary and applying negative voltage, resulting in appearance of both adenosine monophosphate (AMP) and adenosine peaks in less than 2 min. Separations were performed in reverse polarity using -20 kV. Hexadimethrine bromide (HDB) was used as a buffer additive (0.025% in 40 mM borate buffer). An online sample stacking was attained by a special sweeping effect which enabled a long (65 s) injection time without compromise in peak shape. Using this newly developed method, both AMP and adenosine peaks were obtained with a baseline resolution, in less than two minutes. Method was validated by measuring k(m), and V-max values of human ecto-5-nucleotidas and K-i value of standard inhibitor quercitin against this enzyme. Method is simple and fast and can be conveniently optimized for high throughput screening of ecto-5-nucleotidase inhibitors.