The selectivity advantage of MS/MS technology alone is not always sufficient to develop the most suitable method to successfully analyze biological samples. The need for extremely low limits of quantification in bioanalysis requires strong coordination of sample preparation, chromatographic separation and MS/MS conditions. The use of a novel derivatization, two-dimensional chromatography and specialized internal standard are three techniques used to provide robust bioanalytical data.Several different approaches used in the development of methods for tobacco related compounds will be presented.The low limit of quantification of 50 fg/mL for 3-hydroxybenzo[a]pyrene (3-OHBAP) in human urine was achieved by combining an aggressive sample cleanup utilizing the hydrophobic nature of the analyte with derivatization to compensate for the poor efficiency of ionization and mass fragmentation. The combination resulted in a 20-fold increase in sensitivity. The RP UHPLC separation provided additional selectivity to the method as well as increasing signal to noise due to narrow peaks. An elegant two-dimensional chromatography strategy was combining similar column chemistries under different pH conditions was used to provide the needed selectivity to achieve a sub pg/mL limit of quantification for N’-nitrosonornicotine (NNN) at 0.75 pg/mL in human urine. A more traditional two-dimensional LC-MS/MS approach using different types of columns was applied to develop a robust and selective method for monohydroxy-3-butenyl mercapturic acid (MHBMA) in human urine.The fast separation and narrow peak obtained with UHPLC condition may require re-evaluation of the stable internal standard. Carbon-13 labeled internal standards were significantly superior to deuteurated internal standards for mercapturic acids, including hydroxybutyl mercapturic acid (HBMA) and 2-cyanoethylmercapturic acid (CEMA) with respect to compensation for matrix effects and maximizing reproducibility.