Novel, rapid and sensitive kinetic methods and flow injection analysis (FIA) methods with spectrophotometric detection for the determination of N-acetyl-L-cysteine, cysteine, penicillamine, tiopronin and glutathione in their pure form and in pharmaceutical preparations have been developed and validated. The proposed methods are based on the reduction properties of the above mentioned analytes using redox-active colorimetric bis neocuproine copper(II) reagent. The absorbance of the reduced bis neocuproine copper(I) complex is monitored spectrophotometrically at 458 nm. The parameters of the chemical reactions have been optimized. Using kinetic spectrophotometric measurements, the computational initial rate and fixed time methods were utilized for constructing the calibration graphs. For the initial rate method the graphs showed linearity in concentration range from 6 × 10 ̶ 7 mol L ̶ 1 to 8 × 10 ̶ 5 mol L ̶ 1 for N- acetyl-L-cysteine, cysteine and glutathione, and from 8 × 10 ̶ 7 mol L ̶ 1 to 8 × 10 ̶ 5 mol L ̶ 1 for penicillamine and tiopronin, respectively. The slopes of the initial rate method calibration curves (n ≈ 1.0) confirmed the first order reaction for the determination of the above considered thiolic compounds. For the fixed time method the graphs are linear in concentration range from 6 × 10 ̶ 7 mol L ̶ 1 to 8 × 10 ̶ 5 mol L ̶ 1 for N-acetyl-L-cysteine and penicillamine (at 1 min) as well as for cysteine (at 0.5 min), and from 6 × 10 ̶ 7 mol L ̶ 1 to 6 × 10 ̶ 5 mol L ̶ 1 for tiopronin and glutathione (at 1 min), with corresponding limit of detections, equations and correlation coefficients. No interferences were observed from the excipients that are commonly present in pharmaceutical formulations. Both proposed computational methods were successfully applied for the determination of considered thiolic compounds in their commercial pharmaceutical formulations which were compared to the results obtained by the recommended methods according to Britain Pharmacopoeia. During the development of a novel flow injection analysis spectrophotometric procedure for the determination of considered thiolic compounds in pharmaceutical formulations, the primary chemical and flow manifold parameters were examined. Optimum conditions of the parameters are achieved by univariate method. The flow injection methods were validated in terms of linear dynamic range, limit of detection, limit of quantitation, selectivity, precision, and accuracy. The methods found to be linear over the range from 6 × 10 ̶ 7 mol L ̶ 1 to 8 × 10 ̶ 5 mol L ̶ 1 for N-acetyl-L-cysteine and penicillamine, from 6 × 10 ̶ 7 mol L ̶ 1 to 4 × 10 ̶ 5 mol L ̶ 1 for cysteine, from 4 × 10 ̶ 7 mol L ̶ 1 to 8 × 10 ̶ 5 mol L ̶ 1 for tiopronin and from 2 × 10 ̶ 6 mol L ̶ 1 to 8 × 10 ̶ 5 mol L ̶ 1 for glutathione. Measurements confirmed their suitability for the reliable determination of N-acetyl-L-cysteine, cysteine, penicillamine, tiopronin and glutathione in pharmaceutical preparations and dietary supplements at a sampling rate of 180 analyses per hour for N-acetyl-L-cysteine, penicillamine, glutathione and 120 analyses per hour for cysteine and glutathione, respectively. The proposed methods are simple, rapid, sensitive and reproducible with minimal reagent consumption and waste generation.