A direct, ambient ionization method has been developed for the determination of creatinine in urine that combines derivatization and thermal desorption with extractive electrospray ionization and ion mobility-mass spectrometry. The volatility of creatinine was enhanced by a rapid on-probe aqueous acylation reaction, using a custom-made thermal desorption probe, allowing thermal desorption and ionization of the monoacylated derivative. The monoacyl creatinine [M + H](+) ion (m/z 156) was subjected to mass-to-charge selection and collision induced dissociation to remove the acyl group, generating the protonated creatinine [M + H](+) product ion at m/z 114 before an ion mobility separation was applied to reduce chemical noise. Stable isotope dilution using creatinine-d3 as internal standard was used for quantitative measurements. The direct on-probe derivatization allows high sample throughput with a typical cycle time of 1 min per sample. The method shows good linearity (R(2) = 0.986) and repeatability (%RSD 8-10%) in the range of 0.25-2.0 mg/mL. The creatinine concentrations in diluted urine samples from a healthy individual were determined to contain a mean concentration of 1.44 mg/mL creatinine with a precision (%RSD) of 9.9%. The reactive ambient ionization approach demonstrated here has potential for the determination of involatile analytes in urine and other biofluids.