The aim of this work was to generate a tunable surface isoelectric point (sIEP), where the surface is modified with two molecules: a weak base (pyridine), carrying a pH dependent positive charge, and a derivative of a strong acid (sulfate), carrying a permanent negative charge in a physiologically relevant pH range. To this end, silicon surfaces were modified with 3-aminopropyltriethoxysilane. These amine-modified surfaces were subsequently derivatized into pyridine- or sulfate-modified surfaces. Then, the surface pKa of pyridine-modified surfaces was determined by a fluorescent nanoparticle adhesion assay (FNAA). Next, these values were used to calculate in which ratio the chemicals must be present in the reaction mixture to generate a mixed pyridine/sulfate-modified surface with a target sIEP. After preparing surfaces with a target sIEP, an FNAA with positively and negatively charged nanoparticles was used to verify the sIEP of the generated surfaces. The FNAA revealed that pyridine-modified surfaces had a pKa of 6.69 ± 0.18. When an sIEP was generated, negative nanoparticles bound to surfaces at pH values below the sIEP and positive nanoparticles bound at pH values above the sIEP. Furthermore, we found sIEP values of 5.97 ± 0.88 when we aimed for an sIEP of 6.2, and 7.12 ± 0.21 when we aimed for an sIEP of 7.1. Finally, the pH dependent binding and release of a negatively and positively charged (bio)polymer was investigated for a target sIEP of 7. A negatively charged polymer (poly(I:C)) was bound at a pH < sIEP and released at a pH > sIEP with a release efficiency of 85 ± 9% and a positively charged polymer (trimethyl chitosan) bound at a pH > sIEP and released at a pH < sIEP with a release efficiency of 72 ± 9%. In conclusion, we established a method for preparing modified silicon surfaces with a tunable sIEP, which can be used for pH-dependent binding and release of biomacromolecules.