Surface-enhanced Raman spectroscopy (SERS) has recently emerged as an innovative tool for therapeutic-drug monitoring (TDM), making it an ideal candidate for personalized treatment. Herein, we report a layer-by-layer (LbL) approach for the fabrication of a highly reproducible hybrid SERS substrate based on graphene oxide (GO)-supported l-cysteine-functionalized starlike gold nanoparticles (SAuNPs). These designed substrates were utilized for TDM of paclitaxel and cyclophosphamide in blood serum. The SAuNPs' efficient binding at the edges of GO creates a better SERS hotspot with enhanced Raman sensitivity because of the spacing of ∼2.28 nm between the SAuNPs. In addition, the hierarchically modified substrate with a self-assembled monolayer of zwitterionic amino acid l-cysteines acts like a brush layer to prevent SERS-hotspot blockages and fouling by blood-serum proteins. The antifouling nature of the substrate was determined quantitatively by a bichinchonic acid assay using bovine-serum albumin (BSA) as a protein model on the l-cysteine SAuNPs@GO hybrid substrate (the test) and a cysteamine SAuNPs@GO substrate (the control). The l-cysteine SAuNPs@GO hybrid exhibited 80.57% lower BSA fouling compared with that of the cysteamine SAuNPs@GO substrate. The SERS spectra were acquired within 20 s, with detection limits of 1.5 × 10-8 M for paclitaxel and 5 × 10-9 M for cyclophosphamide in blood serum. Such sensitivities are 4 times and 1 order of magnitude higher than the currently available sophisticated analytical techniques, which involve high costs with each analysis.