Background: MYC is a pleiotropic transcription factor aberrantly expressed in up to 70% of human cancers. OMO-103 (Omomyc) is a 91-amino-acids-long polypeptide that derives from the B-HLH-LZ domain of human MYC, containing four amino acid substitutions. It is capable of forming homos and heterodimers with MYC and with MAX and blunt MYC activity by preventing its access to target DNA sequences (E-boxes) and replacing it by inactive complexes. Previous in vivo preclinical experiments showed that Omomyc blocks MYC-driven transcription and proliferation, induces apoptosis and recruitment of intratumoral immune infiltrates, significantly abrogating tumor progression and reducing tumor grading. Here, we present PK results from the first-in-human trial (NCT04808362) of OMO-103 in patients with advanced solid tumors. Methods: Patient eligibility The study included histologically or cytologically proven advanced solid tumors for which there was no curative therapy. Patients had to demonstrate measurable disease according to RECIST v.1.1 criteria as demonstrated by CT/magnetic resonance imaging and documented progression on or following the last line of therapy. Description and dose selection OMO-103 was administered by weekly I.V. infusion over 30–45 min, one treatment cycle corresponding to three infusions. Patients were treated until progression. The starting dose of 0.48 mg kg−1 was determined based on nonclinical toxicology and efficacy studies (anticipated pharmacologically active dose) and subsequent conversion to a human equivalent dose. Pharmacokinetic assays A targeted mass spectrometry (MS) assay for the absolute quantification of 4 Omomyc proteotypic peptides was developed to quantify functional OMO-103 in tumor tissue. LLOQ and linear range of the panel was determined by spiking in dilution series of recombinant OMO-103 protein into tumor tissue lysates. Finally, in the phase I trial, we used the panel for the quantification of OMO-103 in FFPE tumor biopsies from 12 patients who received i.v. injection of OMO-103. Results: MS Data show that 2 h after i.v. administration, higher concentrations of the drug are reached in the tumors compared with serum and persist there with at least one order of magnitude higher concentration than in serum 72 h after administration in preclinical models. Functional Omomyc protein could be quantified in all 12 clinical tumor biopsies from treated patients and PK results confirmed previous observations in preclinical models. where Omomyc exhibits higher tissue concentration and prolonged residency compared to plasma. Conclusions: We demonstrate the utility of targeted MS assay for precise quantification of therapeutic (mini)proteins in clinical tissue samples. Thanks to its multiplexing capability, up to 60 peptides (epitopes) can be monitored simultaneously which enables high sequence coverage and profiling of additional PD or other supporting biomarkers. Clinical trial information: NCT04808362.