Quantitative surface enhanced Raman spectroscopy (SERS) requires precise control of Raman enhancement factor and detection uniformity across the SERS substrate. Here, we show that alkanethiolate ligand-regulated silver (Ag) nanoparticle films can be used to achieve quantitative SERS measurements down to the single-molecule level. The two-dimensional hexagonal close-packed superlattices of Ag nanoparticles formed in these films allow for SERS detection over a large area with excellent uniformity and high Raman enhancement factor. In particular, the SERS signal from the thiolate ligands on Ag nanoparticle surfaces can be utilized as a stable internal calibration standard for reproducible quantitative measurements. We demonstrate the capability of quantitative SERS by measuring the areal densities of crystal violet molecules embedded in an ultrathin spin-on-glass detection "hot zone", which is a planar and uniformly enhanced region several nanometers above the Ag nanoparticles. The Raman measurement results exhibit a linear response over a wide dynamic range of analyte concentration.