Sphingolipids are essential structural and signaling lipids that support membrane integrity and govern cell fate decisions. Whereas the consequences of chronic sphingolipid inhibition have been extensively explored, the immediate cellular responses to reduced sphingolipid levels remain poorly defined. Here, we performed mass spectrometry analysis of subcellular fractions to examine proteome alterations 4 hours after myriocin treatment, which inhibits de novo sphingolipid synthesis. We have identified changes in the abundance of 1307 proteins across the membrane, cytosolic, and nuclear fractions.