The sphingosine 1-phosphate (S1P) signalling pathway controls the neuroinflammatory responses and cellular homeostasis is dependent on the sphingosine kinases SPHK1 and SPHK2. Understanding the function of this pathway in different inflammatory diseases requires an understanding of the correlation of regulatory mechanisms that control it. Using the human cell lines (U251 and HEK 293) this study aims to investigate the impact of six pharmacological agents on the SPHK/S1P signalling pathway. The drugs included in this study were Benzamil HCl, Oxiracetam, PF-543, Resveratrol, Piceatannol, and RO 90-7501. The principal aim was to examine the effects of these drugs on S1P protein concentration, SPHK1 protein/gene expression and SPHK2 gene expression and correlate the relationship between the kinases and S1P concentration. The study explored the functional implications of sphingosine kinase modulation by measuring gene expression and protein levels using quantitative Real-Time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques respectively. The findings highlighted the intricacy of sphingosine metabolism by reflecting that each harmacological agent have unique effects on the SPHK1/SPHK2/S1P axis. In U251 cells treated with Benzamil HCl the SPHK1 gene expression was significantly upregulated however, SPHK1 protein levels and S1P concentrations decreased concurrently indicating the activation of a compensatory feedback loop. Oxiracetam on the other hand has in U251 cells demonstrated downregulation in SPHK2 expression while simultaneously raising S1P protein levels. PF-543 which is a selective SPHK1 inhibitor elicited a compensatory increase in SPHK2 expression in response to diminished S1P levels which emphasizes the interconnected regulatory dynamics of these kinases. Additionally, it has been found that both Resveratrol and Piceatannol had complicated regulatory effects on the metabolism of sphingosine. In HEK 293 cells the Resveratrol demonstrated a nuanced modulation of the sphingolipid pathway by raising the SPHK1 expression while concurrently decreasing the S1P levels. In summary this study adds significant detail to our knowledge of the dynamics of sphingosine signalling and emphasizes on the therapeutic potential of focusing on the SPHK1/SPHK2/S1P pathway in inflammatory conditions. The results highlight how crucial it is to consider pharmacological agents’ specificity and dose-dependent effects opening the door for further research that could result in novel treatment approaches for diseases with dysregulated sphingolipid metabolism.