This paper describes the development and validation of a novel, general liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of cyclophosphamide, ifosfamide, irinotecan, etoposide, gemcitabine, carboplatin and pemetrexed concentrations in human plasma. Samples were prepared by two kinds of extraction method and analyzed using a gradient separation over an Atlantis T3-C18 column (2.1 mm×100 mm, 3 μm, Waters). Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.1% formic acid and 10mM ammonium acetate) at a flow rate of 0.25 mL/min. Linear coefficients of correlation were >0.992 for all analytes. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 9.2%, while the accuracy was within ± 10.5%. The mean recovery of all the analytes ranged from 50.0 to 81.0%. This method was successfully applied to clinical samples from cancer patients.