Cruciferous vegetables are characterized by the presence of sulfur-containing secondary plant metabolites known as glucosinolates (GLS). The consumption of cruciferous vegetables such as broccoli, cabbage, rocket salad, and cauliflower has been related to the prevention of non-communicable diseases. Their beneficial effects are attributed to the enzymatic degradation products of GLS, e.g., isothiocyanates and indoles. Owing to these properties, there has been a shift in the last few years towards the research of these compounds and a wide range of methods for their extraction and analytical determination have been developed. The aim of this review is to present the sample preparation and extraction procedures of isothiocyanates and indoles from cruciferous vegetables and the analytical methods for their determination. The majority of the references that have been reviewed are from the last decade. Although efforts towards the application of eco-friendly non-conventional extraction methods have been made, the use of conventional solvent extraction is mainly applied. The major analytical techniques employed for the qualitative and quantitative analysis of isothiocyanates and indoles are high-performance liquid chromatography and gas chromatography coupled with or without mass spectrometry detection. Nevertheless, the analytical determination of isothiocyanates presents several problems due to their instability and the absence of chromophores, making the simultaneous determination of isothiocyanates and indoles a challenging task.