Methods, systems and kits for the simultaneous or sequential analysis of one or more hormones by mass spectrometry are disclosed. The methods require minimal sample size and minimal preparation time. The methods comprise ionizing the hormones and analyzing the hormones by mass spectrometry. In addition, methods, systems and kits for the simultaneous or sequential analysis of free thyroxine (FT4) hormone and free-triiodothyronine (FT3) is disclosed comprising ionization of the FT4 and FT3 hormone in the negative mode using an electrospray source.