Purpose <p>Intramuscular acton prolongatum is most often used as an alternative for intramuscular or intravenous tetracosactide for ACTH stimulation in some countries. Intramuscular administration of acton prolongatum is cumbersome whereas intended intramuscular acton prolongatum or tetracosactide may often turn subcutaneous. Hence, we compared the subcutaneous ACTH-stimulated steroid profiling with those of the intramuscular routes.</p> Methods <p>The study included 60 apparently-healthy adult females aged 18–40 years who were randomised to intramuscular tetracosactide (250 µg), intramuscular acton prolongatum (24 IU), subcutaneous tetracosactide (250 µg), and subcutaneous acton prolongatum (24 IU) groups in (1:1:1:1) ratio. Serum steroid profile consisting of 13 steroids was measured at baseline and 60 minutes after ACTH stimulation by liquid chromatography-tandem mass spectrometry.</p> Results <p>Baseline steroid levels, stimulated steroid levels, and the percentage increase in all the steroids after subcutaneous tetracosactide and acton prolongatum stimulation were comparable to those after intramuscular tetracosactide and acton prolongatum stimulation, respectively.</p> Conclusions <p>Intramuscular acton prolongatum, subcutaneous tetracosactide and subcutaneous acton prolongatum are simpler and reliable alternatives for ACTH-stimulated steroid profiling.</p>