Phosphoinositides (PIs) play a fundamental role in many physiological processes such as cell surface signal transduction, membrane trafficking, cytoskeleton regulation, nuclear events and permeability and transport functions of membranes. Levels of PIs vary under different physiological conditions thus PI profiling may be an important step in elucidation of importance in the progression towards many diseases. Previous methods for PI analysis have several disadvantages in time-constraints, use of radioactive samples and inconsistent results due to lack of sensitivity. Mass spectrometry has previously been utilized to quantify low abundance peptides, metabolites and lipids. Here we propose a novel approach for PI quantitation based on inositol head cleavage coupled to high-performance liquid-mass spectrometry (HPLC-MS) to overcome these issues. First, we highlight the extraction and deacylation of PIs from S. Ceravisiae, followed by purification via reverse phase chromatography with a Sep-Pak. Next, using commercially purchased PI (4,5) P2 standards, we created a tune file which provide the correct conditions sensitive enough to identify prolific ion peaks to be utilized in characterization of biological samples. Using the standard tune file, we successfully identified and quantitated the PI (4,5) P2 in cells lacking INP51 which have 2-4 fold increase in PI (4,5)P(subscript 2) and comparing them to the wild type cells. The methods described may form a basis for further optimization of mass spectral based quantitation of PIs.