A procedure based on liquid chromatography-electrospray ionization mass spectrometry is described for determination of methylphenidate (MPH) and its principal metabolite ritalinic acid (RA) in plasma, urine, oral fluid and sweat using 3,4-methylendioxypropylamphetamine (MDPA) as internal standard. Aliquots of 100microL biological fluids and sweat patch were initially treated with acetonitrile, centrifuged, and clear supernatants evaporated and redissolved in 10mM ammonium acetate. Chromatography was performed on a reversed-phase column using a gradient of 10mM ammonium acetate and acetonitrile as a mobile phase at a flow rate of 1mL/min. Separated analytes were confirmed and quantified by positive electrospray ionization mass spectrometry and selected ion monitoring acquisition mode. Limits of quantifications were 1ng/mL plasma, 1ng/sweat patch, 0.5ng/mL oral fluid and urine for MHF; 1ng/mL plasma and oral fluid, 1ng/sweat patch, 0.5ng/mL urine for RA using 100microL biological fluids or one sweat-patch per assay. Calibration curves were linear over the calibration ranges for both MPH and RA, with r(2)>0.99. At three concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 67.9-90.3% for MPH and 36.3-92.4% for RA in the different biological matrices. This method was applied to therapeutic monitoring of MHP and RA in conventional and non-conventional biological matrices from individuals in drug treatment.