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A new dansylation reaction, where trifluoromethanesulfonic acid is used as catalyst, has been characterized for six ketosteroids by employing experimental design followed by multivariate data analysis. The molar ratio between the steroid and the derivatization reagent was found to be the factor most strongly affecting the reaction. Faster reaction kinetics was achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the dual peaks observed when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti hydrazones formed. We furthermore conclude that the dansylation reaction is subject to alkyl catalysis rather than acid catalysis, since methyl trifluoromethanesulfonate showed a strong catalytic action, while the catalytic action of trifluoromethanesulfonic acid was lower when diluted in other alcohols and disappeared in aprotic solvents. A sensitivity to water in the reaction mixture strengthens the evidence for alkyl catalysis. When optimal experimental conditions were used, derivatization of picomole amounts of ketosteroids could be accomplished in 25 min. Analysis of spiked plasma containing 0.2-2.0 nmol each of progesterone and 3α-hydroxy-5β-pregnan-20-one showed overall recoveries of 69-76% and 40-55%, respectively. The corresponding 3σ detection limits estimated from calibration curve data were 12 and 15 pmol (n = 4, 500 μL injected).