Mushrooms are considered to be one of the main sources of health promoting bioactive compounds, such as ergothioneine and lovastatin. Sometimes, the analysis of these two compounds can cause confusion since different methods are used. The main purpose of this study was to evaluate the ergothioneine and lovastatin content of different types of mushrooms (Agaricus Bisporus, Pleurotus Ostreatus, Pleurotus Citrinopileatus) as well as Pleurotus Citrinopileatus with cultivation substrates from winery (Grape Marc, GM) and olive oil (OL) by – products, using liquid chromatography – mass spectrometry (LC-MS) and Ultraviolent – Visible spectroscopy spectrometry (UV – Vis). The use of UV – Vis method was hindered due to co-absorbance of different constituents thus LC – MS-MS methodologies were developed, optimized and validated having (a) shorter analysis time and (b) higher resolution. Coefficient factors (R²), accuracy and extraction recoveries confirm the reliability of the methods. Among the studied mushrooms, P. Citrinopileatus contained the highest amount of ergothioneine (822.1 ±20.6) whereas Agaricus Bisporus contained the highest amounts of lovastatin (1.39 ±0.014 mg/kg Dry Sample). As far as the different growth substrates of P. Citrinopileatus is concerned, mushroom samples from olive by products contained the highest amount of ergothioneine (884.5 ± 20 mg/kg Dry Sample), while those from grape marcs contained the highest amounts of lovastatin (0.218 ±0.014 mg/ kg Dry Sample). Finally, since by-products can affect ergothioneine and lovastatin biosynthetic pathways, a colleration between tehir bioactive compounds would be an area of investigation.