Patients with rheumatoid arthritis (RA) are eligible for treatment with therapeutic monoclonal antibodies (mAbs) that target tumor necrosis factor α (TNFα), as well as others, such as rituximab (RTX) and tocilizumab (TCZ). Although pharmacokinetic variability and the link between concentration-clinical response of anti-TNFα mAbs have been well-described, little is known about RTX and TCZ. We aimed to evaluate the variability of RTX and TCZ serum concentrations in RA patients treated in second-line and the relationship between RTX/TCZ concentrations and the clinical response. Serum mAb trough concentrations of RA patients treated with RTX (n = 35) or TCZ (n = 46) were determined at week 24 by liquid chromatography-tandem mass spectrometry. The clinical response was assessed at week 24 by the change in the disease activity score in the 28 joints-erythrocyte sedimentation rate from baseline (ΔDAS28-Erythrocyte Sedimentation Rate) and according to the European League Against Rheumatism (EULAR) recommendations. RTX and TCZ trough concentrations were highly variable, with a coefficient of variation of 171.3% for RTX (median [10th-90th percentiles]: <1.0 µg/mL [<1.0–5.1]) and 132.6% for TCZ (median [10th-90th percentiles]: 5.4 µg/mL [<1.0–27.8]). Univariate analysis did not identify any determinants of such variability, except cotreatment with methotrexate, which was associated with lower RTX concentrations (P = 0.03). The response to treatment was not related to the RTX or TCZ trough concentration. RTX and TCZ trough concentrations at 24 weeks were highly variable in RA patients treated in the second line, without any link concentration-clinical response having been demonstrated.