A new method is described for simultaneous determination of 3-methylindole (3MI) and indole in porcine adipose tissue. Sample preparation included liquid–liquid extraction with n-hexane and 75% aqueous acetonitrile. The acetonitrile extracts were analysed by liquid chromatography–mass spectrometry (LC–MS) using atmospheric pressure chemical ionization (APCI) with selective ion monitoring of protonated ions [M + H]+. This method showed excellent linearity over the concentration range tested (from 2 to 500 ng mL−1 for 3MI and from 1 to 500 ng mL−1 for indole) and good accuracy (recovery of 92 ± 10% for 3MI and 91 ± 10% for indole). This new LC–MS method was compared with traditional colorimetric method for 3MI equivalent. Additionally, the correlation between 3MI concentrations in adipose tissue and plasma was studied. The described LC–MS method can be used to quantify 3MI and indole in porcine adipose tissue in various endocrinological or meat science studies.