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In recent years, derivatives of cathinone, a naturally occurring beta-keto phenylethylamine, have entered the illicit drug market. These compounds have been marketed over the internet or in so-called head shops as "legal highs" and have gained popularity among drug users. Numerous fatalities due to the abuse of these drugs in recent years have increased the need for their detection in human blood samples. For detection and determination of 25 designer cathinones and their related ephedrines in blood samples, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed using only 100 µL of blood. The blood was extracted using liquid-liquid extraction with 1 mL of 1-chlorobutane containing 10% of isopropanol. The final extract was analyzed using a Shimadzu 8030 LC-MS-MS system operated in electrospray positive ionization multiple reaction monitoring mode. The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration for all 25 studied analytes was satisfactory from 10-1,000 ng/mL. Accuracy data were within the acceptance interval of ±15% [±20% at the lower limit of quantification (LLOQ)] of the nominal values for all drugs. Within-day (repeatability) and intermediate precision data were within the required limits of 15% relative standard deviation (RSD) (20% RSD at LLOQ).