Introduction . Multicomponent oral drugs containing salbutamol, bromhexine, ambroxol and guaifenesin have a mucolytic, expectorant and bronchodilator effect. The development method for determination substances in biological fluids is a main procedure for performing the analytical part of pharmacokinetic studies and bioequivalence studies of multicomponent drugs. There is no published data of the determination of bromhexine, ambroxol and guaifenesin, but there a lot of published methods for divided determination analytes in a biological fluid. This study presents the development and validation of a method of the determination of salbutamol, bromhexine, ambroxol and guaifenesin in human blood plasma by high performance liquid chromatography with tandem mass spectrometric detection. A sample preparation was perfomed by solid-phase extraction. Deuterated derivatives were used as internal standards. Aim . The aim of the study is to develop a method for the quantitative determination of salbutamol, bromhexine, ambroxol and guaifenesin in human plasma by HPLC with tandem mass spectrometric detection for performing the analytical part of pharmacokinetic studies. Materials and methods . Determination of salbutamol, bromhexine, ambroxol and guaifenesin in human plasma by HPLC with tandem mass spectrometric detection. A sample was prepared using solid-phase extraction. Results and discussion . The method was validated by next validation parameters: selectivity, matrix effect, calibration curve, accuracy, precision, limit of quantification, carry-over and stability. Conclusion . The method of the determination of salbutamol, bromhexine, ambroxol and guaifenesin in human plasma was developed and validated by HPLC-MS/MS. The analytical range of the was 0.1–20 ng/mL in plasma for salbutamol, 0.25–25 ng/mL in plasma for bromhexine, 0.075–3 ng/mL in plasma for ambroxol, and 10–2000 ng/mL in plasma for guaifenesin. Method could be applied to determination of salbutamol, bromhexine, ambroxol and guaifenesin in plasma for PK and BE studies.