An online turbulent flow chromatography method coupled to tandem mass spectrometry (TFC-MS/MS) has been developed within our bioanalytical group, suited to the analysis of mid to late stage discovery compounds. A dual column configuration utilising isocratic focusing of the analyte upon the analytical column maintained an excellent peak shape for a large proportion of compounds encountered and enabled consistent quantitation to sub-nanogram concentrations (<15 pg on column). Furthermore, the low sample injection volume coupled with rapid column washing using basic and acidic mobile phases, has proved advantageous in removing sample carryover and also the overall exposure to biological material; favourable for good system robustness. All the data discussed were generated with a method cycle time of 5 min providing accurate quantitation (acceptance criteria based upon FDA method validation guidelines) with multiple analytes and biological matrices.