Idiopathic normal pressure hydrocephalus (iNPH) is characterized by reversible neurological symptoms due to an impairment in cerebrospinal fluid (CSF) clearance. In these patients, cognitive functions are severely impaired, with a scenario similar to Alzheimer's disease (AD), making the differential diagnosis difficult and highlighting the need of new markers. We analyzed the composition of sphingolipids (SLs) in serum, by combining a single phase extraction with a high-performance thin-layer chromatography (HPTLC) primuline-profiling, and, in CSF, by MALDI profiling and LC-MS. Ceramides and sphingomyelins (SMs) were similar in serum of iNPH and AD patients compared to healthy controls, whereas, in CSF, MALDI profiling indicated that: 1) SM C24:1 is significantly decreased in AD compared to iNPH patients and controls (Kruskal-Wallis p-value < 0.00001); 2) phosphatidylcholine (PC) 36:2 is increased in iNPH patients (p-value < 0.001). LC-MS identified an increasing trend of Cer C24:0 and of a set of SMs in patients with AD, a significant decrease of sphingosine-1-phosphate (S1P) (t-test p-value 0.0325) and an increase of glucosylceramide (GlcCer) C24:0 (p-value 0.0037) in AD compared to iNPH patients. In conclusion CSF PC 36:2, SM C24:1, S1P, and GlcCer can contribute to improve the differential diagnosis of patients with iNPH or AD and foster preventive therapeutic strategies in the early phase of the disease.