Three dimensional printed open port probe (3DP-OPP) and air displacement based liquid handler, were designed and optimized using fused deposition modeling (FDM) and stereolitography (SLA) 3D printing. The performance of the devices were investigated for the analysis of solid and liquid samples with electrospray ionization mass spectrometry (ESI-MS). Direct analysis in less than 1 min and without any sample preparation, enabled detection of pesticides (azoxtystrobin/imazalil) on fruits peel surface and illegal substances (MDMA/MDEA) in home-made pills. Conjunction of OPP in the overspill mode with a customized autosampler, equipped with disposable pipette tips, enables direct quantitative analysis of drugs of abuse in urine and plasma, with minimized carry-over and reduced matrix effect compared to flow injection analysis.