Bis(monoacylglycero)phosphates (BMPs), a distinct class of anionic phospholipids predominantly found in late endosomes and lysosomes, plays a pivotal role in supporting lysosomal functions and maintaining metabolic homeostasis. Dysregulation of BMPs is associated with an array of disorders, notably neurodegenerative diseases. However, the identification and quantitation of BMP remains difficult because of its structural similarity to its isomer, phosphatidylglycerol (PG), thus necessitating robust analytical methods for accurate and reliable BMP profiling. In this study, we present comprehensive liquid chromatography (LC)-tandem mass spectrometry (MS2) methodologies for the precise and systematic analysis of BMP species in biological samples. We detail LC/MS methods for both an untargeted Orbitrap mass spectrometer and a targeted triple quadrupole mass spectrometer. We use differences in hydrophobicity and structure to annotate BMPs and PGs on the basis of retention time and positive-mode MS2 fragmentation patterns, respectively. Because genetic ablation of the BMP synthase CLN5 leads to specific depletion of BMPs but not PGs, lipid extracts from CLN5 knockout and wild-type cells can be compared to confidently annotate BMPs when MS2 data are incomplete. Lipid extraction and preparation of samples for LC/MS takes ~4 h, unattended LC/MS instrument time depends on the number of samples and computer-based data analysis takes ~1 d. Altogether, this approach constitutes a robust method for BMP profiling and annotation, furthering research into health and disease.