Yes, Skyline works great even if you only have biological replicates and no technical replicates.
We would usually say that you have "one replicate sample" instead of saying that you have "no replicates".
There might be some confusion because Skyline basically calls all of your mass spec data files "Replicates".
The reason for this is that in some PRM or SRM experiments it's not possible to collect data for all of the Targets in a single injection and so the sample was injected into the mass spectrometer resulting in a group of data files. The logical group of data files representing a single well on the plate is called a "Replicate" in Skyline.
When you use the "File > Import > Results" menu item to tell Skyline to extract chromatograms from your data files, the first question that Skyline asks you is whether there were multiple injections.
We have a lot of great tutorials here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials
I would recommend the PRM tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_prm
If you want to learn how to compare results from different cohorts of samples, the Group Comparison tutorial is great:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_grouped
Hope this helps,
-- Nick