Purpose <p>Anabolic–androgenic steroids (AAS) enhance athletic performance, giving athletes an unfair advantage and disrupting fair competition. Banned in sports and listed by World Anti-Doping Agency, they require precise detection. This study aimed to develop a method using the transient matrix effect to improve AAS identification in biological samples.</p> Methods <p>Gas chromatography–tandem mass spectrometry (GC–MS/MS) method for determination of AAS samples was developed and validated. Biological samples were prepared using the QuEChERS technique.</p> Results <p>The optimised and validated method enhances AAS signals using high-boiling protectants. It ensures good linearity, low detection limits, and reliable precision. Optimal QuEChERS extraction and multiple reaction monitoring transitions in GC–MS/MS were evaluated, confirming applicability with blood&#xa0;plasma samples. The addition of a protectant to the analysed sample results in several notable effects. High-boiling protectants, such as polyethylene glycol (PEG-400), tetradecanoic acid (C14-COOH), <i>n</i>-tetradecylalcohol (C14-OH), and <i>n</i>-tetradecylamine (C14-NH₂), significantly enhance AAS’s signal in blood plasma. This enhancement, however, is accompanied by a transient matrix effect induced by the protectants. PEG-400 produced the most substantial signal increase, with the response for nandrolone rising by as much as 912%.</p> Conclusions <p>The results demonstrate the potential offered by the utilisation of PEG-400 as a protectant to generate a transient matrix effect. The outcome of this process is an increased analytical signal from AAS in blood plasma, enabling their identification even at trace concentrations. The methodology developed and applied during the study can be used to reduce the detection limit of steroids and thus improve antidoping measures in sport.</p> Graphical Abstract <p></p>