The stable N -acetyl analogues of biologically important 9- O -acetylated b-series gangliosides including 9NAc-GD3, 9NAc-GD2, 9NAc-GD1b, and 9NAc-GT1b were chemoenzymatically synthesized from a GM3 sphingosine. Two chemoenzymatic methods using either 6-azido-6-deoxy- N -acetylmannosamine (ManNAc6N 3 ) as a chemoenzymatic synthon or 6-acetamido-6-deoxy- N -acetylmannosamine (ManNAc6NAc) as an enzymatic precursor for 9-acetamido-9-deoxy- N -acetylneuraminic acid (Neu5Ac9NAc) were developed and compared for the synthesis of 9NAc-GD3. The latter method was found to be more efficient and was used to produce the desired 9- N -acetylated glycosylsphingosines. Furthermore, glycosylsphingosine acylation reaction conditions were improved to obtain target 9- N -acetylated gangliosides in a faster reaction with an easier purification process compared to the previous acylation conditions.