In the present work two analytical methodologies were developed aiming the determination of thalidomide, an important pharmacological compound. The developed spectrofluorimetric and the voltammetric based analytical methodologies were compared in terms of analytical performance. The thalidomide fluorescent and the electrochemical characteristics were studied in order to find experimental conditions for maximum fluorescence in solution and at room temperature and to allow analyte pre- concentration on the mercury electrode. Using the optimized experimental conditions, limits of detection between 10-6 to 10-9 g L-1 were achieved respectively for the spectrofluorimetric method and for the voltammetric method. Dynamic linear ranges between 2 and 4 orders of magnitude were obtained depending on the method utilized and the interferent substances present in the sample. Those parameters of merit were suitable for this proposed analytical problem. The potential interference effect from substances usually used in association with thalidomide, were studied and strategies for the minimization of such interferences were developed. While no interference in the spectrofluorimetric method was observed for tetracycline, the combined use of acidic medium and UV irradiation of the samples was necessary to allow the analyte determination in the presence of sulfanilamide. For the voltammetric method, interferences from tetracycline and sulfanilamide could be compensated by quantifying thalidomide using the analyte addition method. For the determination in biological fluids, the use a solid-liquid extraction on a C18 column was found to be very effective for the analyte separation and elimination of matrix interferences for the spectrofluorimetric method and for the voltammetric method developed for blood serum. For urine samples, a clean-up step using ammonium sulfate was found to be sufficient for the voltammetric determination of thalidomide. The developed methodologies were tested by determining the thalidomide content in a commercial pharmaceutical formulation and in analyte spiked biological fluids using calibration curves (spectrofluorimetric) and analyte addition method (voltammetric). In all cases, the recoveries were between the 96,5 and 107,6 %, within the recovery range considered adequate according to the United States Pharmacopoeia.