High-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were implemented and optimized to separate and identify sophorolipids (SLs) produced by Rhodotorula bogoriensis and Starmerella bombicola. SLs are carbohydrate-based amphiphilic biosurfactants with increased interest in pharmaceutical and environmental areas. Rhodotorula bogoriensis and Starmerella bombicola are mainly producers of respectively C22, and C16 and C18 SLs. Mass fragmentation patterns of SLs produced by both yeasts were investigated by HPLC-ESI-MS/MS in the positive mode for [M+Na](+). Based on the established fragmentation pattern, SLs produced by both yeasts were identified and characterized. A correlation between HPLC-ELSD and HPLC- ESI-MS/MS methods was established and made possible the identification of SLs by the HPLC-ELSD technique. TLC is a common tool for the analysis of SLs mixtures. In this work, TLC scrapped bands were analysed by HPLC-ELSD and HPLC-MS allowing the correlation between R-F values and the identification of sophorolipids by this technique. Identification of monoacetylated and diacetylated C24:0 hydroxy fatty acids sophorolipids produced by Rhodotorula bogoriensis was for the first time accomplished with this study. Although present in lower quantity these longer chain SLs can assume special importance regarding their biological activity and surface active properties.